Application of cas protein, method for detecting target nucleic acid molecule and kit

ABSTRACT

The present invention provides a use of a Cas protein, and a method and a kit for detecting target nucleic acid molecules. The method for detecting target nucleic acid molecules comprises adding a guide RNA, a Cas12a, and a nucleic acid probe into a reaction system containing target nucleic acid molecules to be detected, and detecting it after the reaction is completed.

TECHNICAL FIELD

The present invention belongs to the field of biotechnology, and inparticular, the present invention relates to a method for the detectionof target nucleic acid molecules.

BACKGROUND

Specific detection method of nucleic acids has important values inapplications such as pathogen detection, genetic disease detection andthe like. For pathogen detections, because each pathogenic microorganismhas its unique characteristic nucleic acid sequences, the detection ofnucleic acid molecules for specific species can be developed, also knownas nucleic acid diagnostics (NADs), plays an important role in the fieldof food safety, environmental microbial pollution detection, humanpathogen infections and the like. In another aspect, the NAD methods canalso detect single nucleotide polymorphisms (SNPs) in human beings orother species. Understanding the relationship between genetic variationand biological function at the genomic level may provide anewperspective for modern molecular biology, among which SNPs are closelyrelated to biological function, evolution, disease and the like, thusthe development of techniques for detection and analysis of SNPs isparticularly important.

So far, several NAD methods have been established, most of which arecreated for the detection of specific DNA molecules and some of whichare for RNA molecules. Usually, DNA molecules are very stable anddetection samples can be from a series of complex biological samples,while RNA molecules can be easily degraded and should be treated withcare. In 1970s, restriction endonuclease digestion detection methodswere developed, and then by the development of methods for detection ofspecific nucleic acid molecules such as Southern, Northern and dothybridization. In 1985 when the PCR method became a routine experimentalmethod, it led to exponential progress in molecular biology. Thedetection of specific nucleic acid molecules currently establishedusually requires two steps, the first step being target nucleic acidamplification and the second step being target nucleic acid detection.PCR technique is the first established and currently most commonly usedamplification method. Based on the PCR method, fluorescence labelledprobes are introduced to facilitate realtime detection of theamplification of target (named as Realtime PCR), which is a rapid andhighly sensitive detection method and can be used for quantitativeanalysis. Besides of PCR amplification method, many alternative methodshave been created, such as the ligase chain reaction, the branched DNAamplification, NASBA, SDA, transcription-mediated amplification,loop-mediated isothermal amplification (LAMP), rolling circleamplification (RCA), Recombinase Polymerase Amplification (RPA) and thelike. The advantage of these alternative methods lies in the isothermalcharacteristic, that's to say that the reaction can be simplyaccomplished at one temperature, without the need for thermal cyclinginstruments for PCR. In addition to real-time PCR, which can directlycomplete amplification and detection, among nucleic acid detectionmethods, FISH hybridization technology (Fluorescence in situhybridization), the most commonly used detection method, hybridizes insitu with complementary target sequences by labeling molecular probes.In addition, detection methods such as next-generation sequencingtechnologies and Oxford Nanopore sequencing technologies have also beendeveloped, but these methods usually require expensive experimentalequipment.

The detection of SNPs first also needs to be amplified by PCR and othermethods to obtain enough amounts of fragments containing SNP siteregions for further detection. Common approaches include primerextension, hybridization, ligation and enzymatic cleavage. Aftercompleting the above methods, you need to use specific methods fordetection, such as mass spectrometry detection, fluorescence detection,chemiluminescence detection, etc.

Although many detection methods have been developed as described abovefor nucleic acid detection, in some cases, faster, simpler and cheaperdetection is still an important development direction, such as rapiddetection of pathogens in the wild, rapid detection of drug-sensitiveSNPs, etc. In 2016, Collins et al. developed a rapid and inexpensivemethod for detecting Zika virus based on the characteristics ofCRISPR-Cas9 that specifically recognizes and cleaves target sequences.In 2017, Zhang Feng et al. took advantage of the collateral effect ofCRISPR-Cas13a to build a rapid nucleic acid detection method.“Collateral effect”, i.e., Cas13a binds to specific target RNA and thenrandomly cleaves other non-target RNAs (here, the RNA molecule isdesigned as an RNA fluorescence reporter system), combined withisothermal amplification technology RPA to achieve rapid target RNAdetection; and Zhang Feng team called this detection method as SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing). The SHERLOCKmethod involves the binding of RNA templates, so if you need to detectDNA, DNA should be first transcribed to RNA for detection; in view ofthe instability of RNA, this method will undoubtedly increase thedifficulty of operation.

In 2015, Zhang Feng et al. discovered a new CRISPR-associated proteinendonuclease Cas12a (formerly known as Cpf1), which is an RNA-guidedspecific DNA endonuclease like the commonly used Cas9 protein; butcompared with Cas9, Cas12a has its own characteristics, such as the needfor crRNA to guide the specific cleavage of double-stranded DNA andproduce cohesive ends.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for detectingtarget nucleic acid molecules.

Another object of the present invention is to provide a use of a Casprotein in a method for detecting target nucleic acid molecules.

In a first aspect of the invention, provided is a kit, comprising aguide RNA, a Cas protein, a nucleic acid probe and a buffer.

provided is a method for detecting target nucleic acid molecules, whichcomprises adding a guide RNA, a Cas protein, a nucleic acid probe and abuffer into the reaction system containing the target nucleic acidmolecules to be detected, and then detecting the target nucleic acidmolecules (especially by detection of the fluorescence intensity).

Preferably, the Cas protein is Cas12a or a Cas protein having anactivity similar to the collateral single-stranded DNA cleavage activityof Cas12a.

Preferably, the Cas protein is Cas12a.

The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a,HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.

Preferably, the Cas12a is LbCas12a.

Preferably, the guide RNA refers to an RNA that directs the Cas proteinto specifically bind to the target DNA.

In another preferred embodiment, the nucleic acid probe is asingle-stranded DNA; the single-stranded DNA is preferably afluorescently labelled single-stranded DNA; and the single-stranded DNAis preferably a fluorescent probe labelled with a fluorescent group HEXat the 5′ end and a quenching group BHQ1 at the 3′ end.

In another preferred embodiment, the detection method of the nucleicacid probe is preferably a fluorescence detection method; and thefluorescence detection method is preferably by using a microplate readeror a fluorescence spectrophotometer.

Preferably, the target nucleic acid molecules to be detected in thereaction system of the target nucleic acid molecules to be detected areobtained after amplification.

Preferably, the detection method of the present invention can detectspathogenic microorganisms, gene mutations or specific target DNA.

In another preferred embodiment, the Cas protein comprises Cas12b(C2c1).

In a second aspect of the invention, provided is a use of a Cas proteinin a method for detecting target nucleic acid molecules, or forpreparing a preparation for the detection of target nucleic acidmolecules.

In another preferred embodiment, when the target DNA, the guide RNA andthe Cas protein form a ternary complex, the complex cleaves othersingle-stranded DNA molecules in the system.

Preferably, the guide RNA refers to an RNA that directs the Cas proteinto specifically bind to the target DNA.

In a third aspect of the invention, provided is a kit comprising a guideRNA, a Cas protein and a nucleic acid probe.

In another preferred embodiment, the kit further comprises a buffer.

In a fourth aspect of the invention, provided is a detection system fordetecting target nucleic acid molecules, comprising:

(a) a Cas protein, which is Cas12a or a Cas protein having an activitysimilar to the collateral single-stranded DNA cleavage activity ofCas12a;

(b) a guide RNA, which directs the Cas protein to specifically bind totarget nucleic acid molecules; and

(c) a nucleic acid probe, which is a single-stranded DNA;

Wherein the target nucleic acid molecule is a target DNA.

In another preferred embodiment, the detection system further comprises(d) a buffer.

In another preferred embodiment, the detection system further comprisestarget nucleic acid molecules to be detected.

In another preferred embodiment, the concentration of the target nucleicacid molecules to be detected in the detection system is from 1 to 100copies/μL or 10¹⁵ copies/μL, preferably from 1 to 10 copies/μL, morepreferably from 1 to 5 copies/μL.

In another preferred embodiment, in the detection system, the molarratio of the nucleic acid probe to the target nucleic acid molecule isfrom 10³:1 to 10¹⁴:1, preferably from 10⁴:1 to 10⁷:1.

In another preferred embodiment, the detection site of the targetnucleic acid molecule is located at positions 1-12 downstream of the PAMsequence of the guide RNA.

In another preferred embodiment, the length of the guide RNA is 15-30nt, preferably 15-18 nt.

In another preferred embodiment, the target DNA comprises cDNA.

In another preferred embodiment, the target DNA is selected from thegroup consisting of: single-stranded DNA, double-stranded DNA, or acombination thereof.

In another preferred embodiment, the nucleic acid probe has afluorescent group and a quenching group.

In another preferred embodiment, the fluorescent group and the quenchinggroup are each independently located at the 5′ end, the 3′ end, and themiddle of the nucleic acid probe.

In another preferred embodiment, the length of the nucleic acid probe isfrom 3 to 300 nt, preferably from 5 to 100 nt, more preferably from 6 to50 nt, and most preferably from 8 to 20 nt.

In another preferred embodiment, the target nucleic acid moleculescomprise target nucleic acid molecules derived from a species selectedfrom the group consisting of: a plant, an animal, an insect, amicroorganism, a virus, or a combination thereof.

In another preferred embodiment, the target DNA is a artificiallysynthesized or a naturally occurring DNA.

In another preferred embodiment, the target DNA comprises a wild-type ora mutant DNA.

In another preferred embodiment, the target DNA comprises DNA obtainedby RNA reverse transcription or amplification, such as cDNA and thelike.

In another preferred embodiment, the Cas12a is selected from the groupconsisting of: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a,OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combinationthereof more preferably, the Cas12a is LbCas12a.

In another preferred embodiment, the Cas protein having an activitysimilar to the collateral single-stranded DNA cleavage activity ofCas12a is selected from the group consisting of: Cas12b (i.e., C2c1).

In another preferred embodiment, the Cas12b protein is selected from thegroup consisting of: AacCas12b (Alicyclobacillus acidoterrestris),Aac2Cas12b (Alicyclobacillus acidiphilus), AkCas12b (Alicyclobacilluskakegawensis), AmCas12b (Alicyclobacillus macrosporangiidus), AhCas12b(Alicyclobacillus herbarius), and AcCas12b (Alicyclobacilluscontaminans).

In another preferred embodiment, the nucleic acid probe comprises asingle-stranded DNA with a detectable label.

In another preferred embodiment, the single-stranded DNA is asingle-stranded DNA labelled with fluorescence and biotin.

In another preferred embodiment, the single-stranded DNA is asingle-stranded DNA labelled with fluorescence.

In another preferred embodiment, the single-stranded DNA is afluorescent probe labelled with a fluorescent group HEX at the 5′ endand a quenching group BHQ1 at the 3′ end.

In a fifth aspect of the invention, provided is a kit for detectingtarget nucleic acid molecules, comprising:

i) a first container and a Cas protein located in the first container,wherein the Cas protein is Cas12a or a Cas protein having an activitysimilar to the collateral single-stranded DNA cleavage activity ofCas12a;

ii) optionally a second container and a guide RNA located in the secondcontainer, wherein the guide RNA directs the Cas protein to specificallybind to the target nucleic acid molecules;

iii) a third container and a nucleic acid probe located in the thirdcontainer;

iv) optionally a fourth container and a buffer located in the fourthcontainer;

Wherein the target nucleic acid molecule is a target DNA.

In another preferred embodiment, any two, three, or four (or all) of thefirst, second, third, and fourth container may be the same or differentcontainer.

In another preferred embodiment, the nucleic acid probe has afluorescent group and a quenching group.

In a sixth aspect of the invention, provided is a method for detectingwhether target nucleic acid molecules are present in a sample,charcterized by comprising the steps of:

(a) providing a detection system for detecting target nucleic acidmolecules according to the fourth aspect of the invention, and thedetection system further contains a sample to be detected; and

(b) detecting whether the nucleic acid probe in the detection system iscleaved by the Cas protein, wherein the cleavage is a trans-cleavage ofthe collateral single-stranded DNA;

Wherein, if the nucleic acid probe is cleaved by the Cas protein, itindicates that the target nucleic acid molecule is present in thesample; and if the nucleic acid probe is not cleaved by the Cas protein,it indicates that the target nucleic acid molecule is not present in thesample.

In another preferred embodiment, the sample to be detected comprises anunamplified sample and an amplified (or nucleic acid amplified) sample.

In another preferred embodiment, the sample to be detected is a sampleobtained by amplification.

In another preferred embodiment, the nucleic acid amplification methodis selected from the group consisting of: PCR amplification, LAMPamplification, RPA amplification, ligase chain reaction, branched DNAamplification, NASBA, SDA, transcription-mediated amplification, rollingcircle amplification, HDA, SPIA, NEAR, TMA and SMAP2.

In another preferred embodiment, the PCR comprises high temperature PCR,normal temperature PCR, and low temperature PCR.

In another preferred embodiment, the method is for detecting whetherthere is an SNP, a point mutation, a deletion, and/or an insertion fornucleic acids at a target site.

In another preferred embodiment, when the PAM sequence is absent at theupstream or downstream of the target site (in the range of from −20 ntto +20 nt, preferably in the range of from −15 nt to +15 nt, and morepreferably in the range of from −10 nt to +10 nt), nucleic acidamplification was carried out using primers introduced with PAM.

In another preferred embodiment, the primer introduced with PAM has astructure of formula I from 5′ to 3′:

P1-P2-P3  (I)

wherein,

P1 is a 5′ segment sequence complementary or non-complementary to thesequence of the target nucleic acid molecule at the 5′ end;

P2 is a PAM sequence; and

P3 is a 3′ segment sequence complementary to the sequence of the targetnucleic acid molecule at the 3′ end.

In another preferred embodiment, the PAM primer specifically bindsupstream or downstream of the target nucleic acid molecule.

In another preferred embodiment, P1 has a length of 0 to 20 nt.

In another preferred embodiment, P3 has a length of 5 to 20 nt.

In another preferred embodiment, the PAM primer has a length of 18 to 50nt, preferably 20 to 35 nt.

In another preferred embodiment, the complementation comprises fullcomplementation and partial complementation.

In another preferred embodiment, at least one primer with a PAM sequenceis used in the nucleic acid amplification.

In another preferred embodiment, when the PAM sequence is present at theupstream or downstream of the target site (in the range of from −20 ntto +20 nt, preferably in the range of from −15 nt to +15 nt, and morepreferably in the range of from −10 nt to +10 nt), primers with orwithout a PAM sequence can be used, and the amplified amplificationproduct contains the PAM sequence.

In another preferred embodiment, the detection in step (b) comprises afluorescence detection method.

In another preferred embodiment, a microplate reader or a fluorescencespectrophotometer is used in the fluorescence detection method.

In a seventh aspect of the invention, provided is a use of a Cas proteinfor the preparation of a detection reagent or kit for detecting targetnucleic acid molecules based on a collateral single-stranded DNAcleavage, wherein the Cas protein is Cas12a or a Cas protein having anactivity similar to the collateral single-stranded DNA cleavage activityof Cas12a.

In another preferred embodiment, the Cas12a is selected from the groupconsisting of: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a,OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a or a combinationthereof; more preferably, the Cas12a is LbCas12a.

In another preferred embodiment, the Cas protein having an activitysimilar to the collateral single-stranded DNA cleavage activity of Casis selected from the group consisting of: Cas12b (or C2c1).

In another preferred embodiment, the Cas12b protein is selected from thegroup consisting of: AacCas 12b.

It should be understood that within the scope of the present invention,the various technical features of the present invention above and thevarious technical features specifically described hereinafter (as in theExamples) may be combined with each other to constitute a new orpreferred technical solution. Due to space limitations, it is notrepeated here.

DESCRIPTION OF FIGURE

FIG. 1 shows the cis-cleavage characteristics of the Cas12a cleavingtarget single-stranded DNA.

FIG. 2 shows Cas12a cleaves target single-stranded DNA independent ofPAM sequence required for cleaving double strands.

FIG. 3 shows the trans-cleavage characteristics of the Cas12a cleavingsingle-stranded DNA.

FIG. 4 shows 10 different sources of Cas12a tested, all of which havecis- and trans-cleavage activity for single-stranded DNA.

FIG. 5 shows through Cas12a single-point mutation experiment, the sitesin Cas12a that may be related to the cis- and trans- cleavage activityof single-stranded DNA were identified.

FIG. 6 shows the structures of Cas12a and Cas12b (i.e., C2c1) monomersand their complexes with guide RNA and target DNA.

FIG. 7 shows different Cas12a uses specific double-stranded DNAsubstrates and single-stranded DNA (HEX-N12-BHQ1) as the fluorescentdetection probe to obtain the fluorescence value. The negative controlgroup does not add specific substrates. No specific substrate added wasemployed as the negative control group.

FIG. 8 shows schematic of target DNA detection by the HOLMES methodbased on target DNA amplification and Cas12a trans-cleavage activityagainst collateral single-stranded DNA.

FIG. 9 shows sensitivity test of target DNA by direct use of FnCas12a orLbCas12a, or in combination of the HOLMES method.

FIG. 10 shows fluorescence detection value of the HOLMES method usingcrRNAs with different lengths of the guide sequence combined withFnCas12a or LbCas12a on target sequences with different single pointmutations.

FIG. 11 shows using FAM-labeled fluorescent probes, 10 Cas12a proteinswere selected to test whether the FAM-labeled single-stranded DNA probeis trans-cleaved after adding the target single-stranded DNA.

FIG. 12 shows using HEX-N12-BHQ1 as a probe, 10 Cas12a proteins wereselected to test the fluorescence value after adding the targetsingle-stranded DNA.

FIG. 13 . (A) Single-stranded DNA fluorescent probes labeled with HEXand BHQ1 at both ends show HOLMES detection values using the gyrB genefragment as the target sequence and different concentrations of purecultured E. coli MG1655 as the positive control template. It is shownthat as the concentration of E. coli MG1655 decreases, its fluorescenceresponse value gradually decreases. (B) Detection values ofenvironmental water samples in different locations.

FIG. 14 shows schematic diagram of the HOLMES method for detecting SNPs,and the fluorescence detection values of 5 SNP sites.

FIG. 15 shows the fluorescence detection values in key sites when usingHOLMES method for detecting TP53 gene (a cancer-related gene).

FIG. 16 shows the detection values of 5 SNP sites (gout related) byusing HOLMES method.

FIG. 17 shows the detection values of 1 SNP site (gout related) by usingHOLMES method, with samples from 21 volunteers.

FIG. 18 shows a scheme for primers design in one Example of the presentinvention, which could be used for HOLMES detection of SNP in any site.

FIG. 19 shows the detection of E. coli with the combination of LAMP andHOLMES in the system. (A) The electrophoretogram of the E. coli gyrBgene amplified by LAMP. Two groups of primers (i.e. gyrB-1 and gyrB-2)were used to amplify the gyrB gene, which is the informative gene of E.coli. (B) Detection of the LAMP amplification products with HOLMESdetection system. Negative control: The sample was sterilized water, andthe gyrB gene was amplified or the result thereof was detected withgyrB-1 amplification primers. gyrB-1: The sample was the E. coli to bedetected, and gyrB gene was amplified or the result thereof was detectedwith the first group of gyrB gene amplification primers. gyrB-2: Thesample was the E. coli to be detected, and gyrB gene was amplified orthe result thereof was detected with the second group of gyrB geneamplification primers.

FIG. 20 shows the detection of the genotypes of human HEK293T cellsthrough the combination detection of LAMP and HOLMES. (A) Theelectrophoretogram of SNP detection template corresponding to humanHEK293T amplified by LAMP. Negative control: The sample was sterilizedwater, and the amplification result was detected with rs5082amplification primers. rs5082: The sample was the total genomic DNA fromhuman HEK293T cells, and the amplification result was detected withrs5082 amplification primers. rs1467558: The sample was the genomic DNAfrom human HEK293T cells, and the amplification result was detected withrs1467558 amplification primers. (B) Detection of the LAMP amplificationproduct with HOLMES detection system. Two crRNAs, crRNA-G and crRNA-Twere used for detection of the rs5082 site (Sequence Listing 5),respectively, and two crRNAs, crRNA-C and crRNA-T were used fordetection of the rs1467558 site (Sequence Listing 5), respectively.

FIG. 21 shows the detection of E. coli with the combination of RPA andHOLMES in the system. (A) Amplification of the E. coli gyrB gene withRPA. Two groups of primers (i.e. gyrB-1 and gyrB-2) were used to amplifythe gyrB gene, which is the informative gene of E. coli. (B) Detectionof the RPA amplification products with HOLMES detection system. Negativecontrol: The sample was sterilized water, and the gyrB gene wasamplified or the result thereof was detected with gyrB-1 amplificationprimers. gyrB-1: The sample was the E. coli to be detected, and gyrBgene was amplified or the result thereof was detected with the firstgroup of gyrB amplification primers. gyrB-2: The sample was the E. colito be detected, and gyrB gene was amplified or the result thereof wasdetected with the second group of gyrB amplification primers.

FIG. 22 shows the detection of the collateral ssDNA cleavage activity ofCas12b with ssDNA as target DNA. After the collateral cleavage reaction,the reactants were subject to denatured gel electrophoresis separationwith 12% urea and detected with the fluorescence imaging system. Thenumber in the brackets stood for the final concentration, in nM, ofreactants. The 50 nM ssDNA of 66 nts was used as the target DNA, and 50nM 5′-end FAM-labelled ssDNA was used as the ssDNA probe. As shown inthe figure, with the addition of Cas12b, guide RNA and target DNA,FAM-labelled ssDNA was cleaved into fragments, demonstrating that Cas12bhad collateral ssDNA cleavage activity.

FIG. 23 shows the detection of the collateral ssDNA cleavage activity ofCas with ssDNA or dsDNA as target DNA. After collateral cleavagereaction was completed, the reactants were detected with a fluorescencemicroplate reader. The amount of Cas12b and guide RNA was 500 nM. 50 nMssDNA (66-nt) or dsDNA (66-nt) was used as the target DNA. 500 nM ssDNAprobe containing fluorescence reporter and quencher group (HEX-N12-BHQ1)was used as the ssDNA probe. As shown in the figure, upon the additionof Cas12b and guide RNA, no matter ssDNA template or dsDNA template, thecollateral ssDNA cleavage activity could be detected.

FIG. 24 shows the collateral ssDNA trans-cleavage activity generated byCas12b with the combination of LAMP amplification for low-concentrationtarget DNA.

DETAILED DESCRIPTION

In order to make the aim, technical solution and advantages of theExamples of the present invention clearer, the technical solutions inthe embodiments of the present invention will be clearly and completelydescribed in conjunction with the drawings in the Examples of thepresent invention. Obviously, the Examples described herein are not allof the Examples of the present invention. All other Examples obtained bythose skilled in the art based on the Examples of the present inventionwithout creative efforts are within the scope of the present invention.

By extensively and intensively studies, the present inventors havedeveloped a technical solution for target nucleic acid detection, bystudying the cleavage properties of Cas enzymes such as Cas12a andCas12b enzymes. The experimental results show that the above technicalsolutions are successfully used to rapidly detect nucleic acids. Forexample, identification of whether the water contains a certainconcentration of microorganisms such as Escherichia coli and rapididentification of SNP genotypes are accomplished. On this basis, thepresent invention has been completed.

The Terms

The term “guide RNA” refers to an RNA that directs the Cas protein tospecifically bind to a target DNA sequence.

The term “crRNA” refers to CRISPR RNA, which is a short RNA that directsCas12a to bind to a target DNA sequence.

The term “CRISPR” refers to clustered regular interspaced shortpalindromic repeats, which are the immune systems of many prokaryotes.

The term “Cas protein” refers to a CRISPR-associated protein which is arelated protein in the CRISPR system.

The term “Cas12a” (formerly “Cpf1”) refers to a crRNA-dependentendonuclease, which is an enzyme of type V-A in the CRISPR systemclassification.

The terms “Cas12b”, and “C2c1” are used interchangeably and refer to acrRNA-dependent endonuclease, which is an enzyme of type V-B in theCRISPR system classification.

The term “LAMP” is a loop-mediated isothermal amplification techniqueand is a thermostatic nucleic acid amplification technique suitable forgene diagnosis.

The term “PAM” refers to a protospacer-adjacent motif, which is requiredfor Cas12a cleavage. The PAM of FnCas12a is the sequence TTN, the PAM ofLbCas12a is the sequence TTTN, and the PAM of AacCas12b is the sequenceTTN.

The present invention provides a method for detecting target nucleicacid molecules, which comprises adding a guide RNA, a Cas protein, anucleic acid probe and a buffer into the reaction system containing thetarget nucleic acid molecules to be detected, and then detecting thefluorescence intensity thereof.

The Cas protein is Cas12a or Cas12b.

The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a,HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; theCas12a is preferably LbCas12a.

The Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b,AhCas12b, and AcCas 12b.

The guide RNA refers to an RNA that directs the Cas protein tospecifically target a DNA sequence.

The target nucleic acid molecules to be detected in the reaction systemcontaining the target nucleic acid molecules to be detected are obtainedafter amplification.

The detection method detects pathogenic microorganisms, gene mutationsor specific target DNA.

The present invention provides a use of a Cas protein in a method fordetecting target nucleic acid molecules.

When the target DNA, the guide RNA and the Cas protein form a ternarycomplex, the complex cleaves other single-stranded DNA molecules in thesystem.

The guide RNA refers to an RNA that directs the Cas protein tospecifically target a DNA sequence.

The invention also provides a kit, comprising a guide RNA, a Cas proteinand a nucleic acid probe. Furthermore, the kit of the invention mayfurther comprise a buffer.

The present invention provides a detection method for rapidly detectingtarget nucleic acid molecules with high specificity. Once the target DNA(single or double stranded), the crRNA, and the Cas12a protein form aternary complex, the complex cleaves other single-stranded DNA moleculesin the system. In the method, the target DNA (a DNA sequence to bedetected) is targeted by the designed crRNA; and crRNA and Cas12aprotein are added to the detection system. When the target DNA ispresent, Cas12a, the crRNA and the target DNA form a ternary complex,and the complex cleaves the single-stranded DNA with fluorescent signallabel (the fluorescent group and the quenching group are respectivelylabelled to the two ends, and the fluorescent group can be luminescentafter the DNA is cleaved) using its collateral cleavage activity,thereby emitting fluorescence. Therefore, by detecting the fluorescence,it is possible to know whether or not the target DNA molecule iscontained in the system to be detected. The method of the presentinvention allows rapid detection of whether a sample contains a specificDNA sequence. By combining with PCR technology, the sensitivity of thedetection method can be greatly improved. The nucleic acid probe in thepresent invention is preferably a fluorescent probe.

HOLMES Condition Test

The invention provides the application of Cas12 enzyme based on Cas12a,Cas12b and the like in nucleic acid detection. The following describesCas12a as an example.

Selection of Cas12a: According to the study, Cas12a has the activity oftrans cleavage, ie, once the target DNA, crRNA and Cas12a protein form aternary complex, other single-stranded DNA (collateral single-strandedDNA) in the system will be cleaved. A specific DNA detection method wasdesigned based on this principle. Firstly, the collateral DNA wasdesigned as a fluorescent probe consisting of a random sequence with alength of 12 nt, and labelled with the fluorescent group HEX at the5′-terminal end and labelled with the quenching group BHQ1(HEX-N12-BHQ1) at the 3′-terminal end. When the system contains thetarget DNA fragment, a ternary complex of the target DNA, crRNA andCas12a protein will be formed. At this time, the probe is cleaved, andthe fluorescence emitted from the HEX fluorescent group can be detectedby the fluorescence detector (excitation at 535 nM, and emission at 556nM). Secondly, 10 different Cas12a were tested, and the target sequencewas double-stranded DNA, as shown in FIG. 7 . It can be seen that thecomplex composed of the target double-stranded DNA and each Cas12aprotein can achieve trans cleavage activity.

HOLMES Response Sensitivity: Next, the sensitivities of FnCas12a andLbCas12a response to target DNA were tested. That is, the lowestconcentration of target DNA that can respond was examined. As shown inFIG. 9 , when the test target was directly added, the target DNA withconcentration above 0.1 nM was able to respond, and the response wassignificant at a concentration above 1 nM. If the PCR technique iscombined (i.e. HOLMES method), as shown in FIG. 8 , i.e. the targetfragment is first amplified by PCR and then subjected to Cas12a cleavagereaction, the response sensitivity can be as low as 10 aM, as shown inFIG. 9 .

SNP test: Next, whether the HOLMES method can detect the SNP genotypewas tested. T1 was used as the target sequence, and mutations wereintroduced in the PAM or single point mutation was introduced in thetarget sequence at position of 1-18. And the difference between thenon-mutated sequence and the mutant sequences was compared for thecrRNAs of different lengths.

As shown in FIG. 10 , when the target complementary sequence is a 24 ntcrRNA (crRNA-24 nt), the single point mutations at position of 8-18 arenot significantly different from the wild type. When there is anymutation in the PAM and there is any point mutation at position of 1-7,there is a significant drop on the fluorescence value. When the crRNAwas truncated and the length of the paired target sequence was 18 nt,the fluorescence value of the target sequence mutated at position 8-16nt was significantly lower than that of the target sequence with alength of 24 nt. When the length was shortened to 16 nt or 17 nt, thedecrease of fluorescence value of the target sequence after mutation wasmore obvious. When the length was shortened to 15 nt, the fluorescencevalues of the target sequence and the mutation are weak for the targetsequence, but still strong for other target sequences, which can be usedfor detection. Taken together, 15 nt, 16 nt and 17 nt crRNAs are mostsuitable for detection of SNPs.

In the present invention, Cas12a cleaves single-stranded DNA. Aprogrammed cleavage mode wherein Cas12a cleaves single-stranded DNAindependent of PAM sequence is called cis cleavage; and once a ternarycomplex Cas12a/crRNA/target DNA is formed, it shows the activity oftrans-cleavage, that is, the activity of cleavage of any non-targetsingle-stranded DNA in the system.

Using the characteristics of Cas12a, a method for specifically detectingnucleic acid molecules has been developed, which is called HOLMES (oneHour Low-cost Multipurpose Efficient Simple assay). As the name of thetechnology, it is characterized as a fast (1 hour), low cost,multi-purpose, efficient, and simple method. The method can be used inthe fields of rapid pathogen detection, SNP detection and the like.

Nucleic Acid Detection Based on Collateral Cleavage Activity

The invention also provides a method of nucleic acid detection based onthe collateral cleavage activity of a Cas12 enzyme, including Cas12a orCas12b.

Preferably, the detection of the invention can be performed on SNPs, inparticular by PCR amplification followed by detection.

Referring to FIG. 18 , the design of the primers is given.

Case 1. When there is a PAM site near the SNP site, the synthesizedcrRNA based on the guide sequence designed according to the PAM site canbe used for HOLMES detection. When the HOLMES method is used fordetection, the background signal is relatively low; for the same guidesequence, the signal difference between different SNP templates isrelatively large.

Case 2. When there is no PAM site near the SNP site, or there is nosuitable PAM site, the introduction of the PAM site can be performedaccording to the above experimental protocol.

A typical procedure comprises designing a primer near the SNP site,carrying a PAM site on the primer, and the 3′ end sequence at the PAMsite needs to be paired with the template DNA. There is no specialrequirements for the primer at the other end, and it only needs to bepaired with the template DNA and can be subjected to PCR amplification.As shown in FIG. 18 , the PAM site was successfully introduced after PCRamplification.

Referring to FIG. 10 , in the present invention, when designing for theintroduction of a PAM, usually the SNP site is located at the positionof the first 16 bases of from 5′ end of the crRNA guide sequence,preferably 1-14, more preferably 1-12, more preferably 1-11 or 1-10, andmost preferably 1-8 or 1-7.

The main advantages of the invention are:

(1) Fast: When the test conditions are ready, it takes only about 1 hourfrom getting the sample to getting the test result.

(2) Low cost: There are no special materials or enzymes required in theexperiment, and the amount of materials and reagents involved is small.It can be used for testing and analysis of trace amounts.

(3) Efficient: The method of the present invention has extremely highsensitivity and can detect DNA at a concentration of 10 aM.

(4) Multi-purpose: It can detect different nucleic acid samples,including DNA samples and RNA samples.

(5) Simple: There are no special complicated steps. If the kit is readyand the program is set, only the steps of simply adding the sample andthe like are needed.

The invention is further illustrated below in conjunction with specificExamples. It should be understood that the Examples are provided toillustrate the present invention, but are not intended to limit thescope of the invention. The experimental methods in the followingExamples which do not specify the specific conditions are usually inaccordance with conventional conditions, such as conditions described inSambrook et al., Molecular Cloning: Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989), or in accordance with theconditions recommended by the manufacturer. Unless otherwise stated,percentages and parts are by weight.

The experimental materials involved in the present invention can beobtained commercially unless otherwise specified.

Materials

1. The RNase inhibitor was purchased from TaKaRa. The high-fidelity DNApolymerase KOD FX was purchased from ToYoBo. The primers(oligonucleotides) were synthesized by Shanghai Sangon Biotech. The T7RNA polymerase was purchased from Thermo. The RNA purification andconcentration kit (RNA Clean & Concentrator™-5) was purchased from ZymoResearch. Wizard® SV Gel and PCR Clean-Up System was purchased fromPromega. The media (e.g. Tryptone, Yeast Extract, etc.) were purchasedfrom OXOID.

2. Medium formula: liquid LB (1% Tryptone, 0.5% Yeast extract, 1% NaCl).When formulating solid LB, only 2% agar should be added to the liquidLB.

Example 1. Detection of Detectable Single-Stranded DNA Target by Cas12aProteins (FAM-Labelled Probe).

Single-stranded DNA (target-T1-R) was used as the target sequence totest the response values of different Cas12a proteins for detectionthereof.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-T1-24-R, as shown in table 5, were annealed toprepare the template for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×PCR buffer (Transgen. Biotech.) witha volume of 50-μL, following the annealing program: initial denaturationat 95° C. for 5 min, then cooling down from 95° C. to 20° C. usingthermocycler (decrease by 1° C. per min). crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). Then, RNA is purified using RNA purification andconcentration kits, quantitated with NanoDrop 2000C (Thermo FisherScientific), diluted to a concentration of 10 μM and stored at −80° C.in a refrigerator.

2. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM Cas12a, 0.01 μM targetsingle-stranded DNA (target-T1-R), 0.01 μM nucleic acid probe (N25-5′FAM), NEB buffer 3.1, and 0.5 μL of RNA enzyme inhibitor. For blankcontrol reaction, all other components were added except single-strandedDNA target sequence. The reaction was carried out at 37° C. for 15 min,and then stopped at 98° C. for 2 min.

3. Fluorescence detection: Electrophoresis was carried out withurea-polyacrylamide gel electrophoresis (Urea-PAGE) and then detectionwas carried out with the fluorescence luminescence imager. As shown inFIG. 11 , different Cas12as showed distinct target defection effects.For example, HkCas12a and the like cleaved the probe even without targetsingle-stranded DNA being added. However, LbCas12a and the like cleavedthe probe only with target single-stranded DNA being added, and weregood Cas12a protein candidates.

Example 2. Detection of Detectable Single-Stranded DNA Target by Cas12aProteins (Dural HEX & BHQ1-Labelled Probes).

Single-stranded DNA (target-T1-R) was used as the target sequence totest the response values of different Cas proteins for detectionthereof.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-T1-24-R (Table 5) were annealed and used toprepare the template for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×PCR buffer (Transgen Biotech) with avolume of 50-μL, following the annealing procedure: initial denaturationat 95° C. for 5 min, and then cooling down from 95° C. to 20° C. usingthermocycler (decrease by 1° C. per min). crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). Then, RNA was purified with an RNA purificationand concentration kit, quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM Cas12a, 0.01 μM targetsingle-stranded DNA (target-T1-R), 0.5 μM fluorescent probe(HEX-N12-BHQ1. i.e. 12 nt single-stranded DNA labelled with HEX at the5′ end and BHQ1 at the 3′ end), NEB buffer 3.1, and 0.5 μL of RNA enzymeinhibitor. For control reaction, all other components were added exceptsingle-stranded DNA target sequence. The reaction was carried out at 37°C. for 15 min, and then stopped at 98° C. for 2 min.

3. Fluorescence detection: 20 μL, of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.12 , different Cas12as showed distinct target defection effects. Forexample, HkCas12a and the like cleaved the probe even without targetsingle-stranded DNA being added. However, FnCas12a and the like cleavedthe probe only with target single-stranded DNA being added, and weregood Cas12a protein candidates.

Example 3. Detection of Detectable Double-Stranded DNA Target by Cas12aProteins

Double-stranded DNA (target-T1) was used as the target sequence to testthe response values of different Cas proteins for detection thereof.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-T1-24-R (Table 5) were annealed to prepare thetemplate for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×PCR buffer (Transgen Biotech) with avolume of 50-μL, following the annealing procedure: initial denaturationat 95° C. for 5 min, and then cooling down from 95° C. to 20° C. usingthermocycler (decrease by 1° C. per min). crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). Then, RNA was purified with an RNA purificationand concentration kit, quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM Cas12a, 0.01 μM targetdouble-stranded DNA (target-T1, obtained from annealing of primertarget-T1-F and primer target-T1-R), 0.5 μM fluorescent probe(HEX-N12-BHQ1), NEB buffer 3.1, and 0.5 μL of RNA enzyme inhibitor. Thereaction was carried out at 37° C. for 15 min, and then stopped at 98°C. for 2 min.

3. Fluorescence detection: 20 μL, of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG. 7, different Cas12as showed distinct target defection effects. WhereasLbCas12a and the like cleaved the probe only with target double-strandedDNA being added, and were good Cas12a protein candidates.

Example 4. Testing of Targets at Different Concentrations by FnCas12aand LbCas12a

Target-T1 DNA was used as target DNA, then subjected to gradientdilution into different concentrations to test the sensitivity ofFnCas12a or LbCas12a response to it. To enhance the sensitivity, PCRamplification step was added.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-T1-24-R (Table 5) were annealed to prepare thetemplate for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×PCR buffer (Transgen Biotech) with avolume of 50-μL, following the annealing procedure: initial denaturationat 95° C. for 5 min, and then cooling down from 95° C. to 20° C. usingthermocycler (decrease by 1° C. per min). crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). The RNA was purified with an RNA purificationand concentration kit, quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. PCR amplification (optional): Plasmid (pUC18-T1) that contained thetarget-T1 target, as the template, was subjected to gradient dilutionand PCR reaction. The total volume of each reaction system was 20 μL,0.25 μM of primers of M13F-47 and M13R-48 ware used (Table 4), andhigh-fidelity enzyme KOD FX (ToYoBo) was used for PCR reaction. The PCRreaction procedure was at 95° C. for 2 min, followed by 35 cycles of 98°C. for 10 s, 60° C. for 15s and 68° C. for 10 s. After PCR wascompleted, the PCR purification products were directly used for Cas12areaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM FnCas12a or LbCas12a, 1 μL of PCRproducts (or target DNA directly diluted to different concentrations),0.5 μM fluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1, and 0.5 μL ofRNA enzyme inhibitor. The reaction was carried out at 37° C. for 15 min,and then stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG. 9, target DNA at or above 0.1 nM was able to response when the testtargets were directly added, and response was significant when theconcentration was above 1 nM. However, combined with PCR technology,i.e., first PCR amplification of fragments of interest and then Cas12acleavage reaction, the response sensitivity could be as low as 10 aM.

Example 5. Testing of Single-Point Mutation Target by FnCas12a andLbCas12a.

Target-T1 DNA was used as the target and mutated in the PAM region andposition 1-18, respectively. Several crRNAs of different lengths weretested for response values to the wild type and single-point mutatedtargets.

1. Preparation of crRNA: First, T7-crRNA-F was annealed with syntheticoligonucleotides of T7-T1-24-R, T7-T1-15-R, T7-T1-16-R, T7-T1-17-R andT7-T1-18-R (Table 5), respectively, to prepare templates fortranscription. Specifically, 4 μM of paired oligonucleotides wereannealed in 1×PCR buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). crRNA was synthesized using T7 high-yieldtranscription kit, and the reaction was performed at 37° C. overnight(about 16 h). The RNA was purified with an RNA purification andconcentration kit, quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. PCR amplification: Plasmid (pUC18-T1) that contained the target-T1target was used as the template. The total volume of each reactionsystem was 20 μL, 0.25 μM primers of M13R-48 and each mutant primer forTarget-T1-F were used (Table 4), and high-fidelity enzyme KOD FX(ToYoBo) was used for PCR reaction. The PCR reaction procedure was at95° C. for 2 min, followed by 35 cycles of 98° C. for 10 s, 60° C. for15 s and 68° C. for 10 s. After PCR was completed, they were directlyused for Cas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM FnCas12a or LbCas12a, 1 μL of PCRproducts, 0.5 μM fluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1, and0.5 μL of RNA enzyme inhibitor. The reaction was carried out at 37° C.for 15 min, and then stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.10 , when the target complementary sequence was 24 nt crRNA (crRNA-24nt), no significant difference was observed between single-point mutatedtarget with mutation at position 8-18 and the wild type, and aftermutation within PAM and at position 1-7, the fluorescence value wasobviously decreased. When crRNAs were truncated and the paired targetsequence length was 18 nt, the fluorescence value of the mutation atposition 8-16 nt was remarkably lower than that at 24 nt. At 16 nt or 17nt, the fluorescence value of the mutated target sequence was decreasedmore remarkably. At 15 nt, the fluorescence value of both the targetsequences and the mutants was very weak. However, the strength thereofcould be still high for other target sequences, which could be used fordetection. To sum up, crRNAs of 15 nt, 16 nt and 17 nt were the mostsuitable for detection of SNP.

Example 6. Testing of Microorganisms Such as E. coli in EnvironmentalWater

The E. coli gyrB gene was selected as the detection target to indirectlytest the concentration of microorganisms such as E. coli in the water.E. coli MG1655 was used as the positive control to determine the contentof microorganisms in the environmental water such as polluted water andrunning water.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-crRNA-gyrB (Table 5) were annealed to prepare thetemplate for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×PCR buffer (Transgen Biotech) with avolume of 50-μL, following the annealing procedure: initial denaturationat 95° C. for 5 min, and then cooling down from 95° C. to 20° C. usingthermocycler (decrease by 1° C. per min). crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). The RNA was purified with an RNA purificationand concentration kit, quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. PCR amplification: The positive control sample was E. coli MG1655which was cultured till the OD₆₀₀ reached about 0.5, then subjected to10 times gradient dilution and used as the template. Samples were takenfrom environmental water, including running water and environmental mudwater. The total volume of each reaction system was 20 μL, 0.25 μMprimers of gyrB-F and gyrB-R were used (Table 4), and high-fidelityenzyme KOD FX (ToYoBo) was used for PCR reaction. The PCR reactionprocedure was at 95° C. for 2 min, followed by 35 cycles of 98° C. for10 s, 60° C. for 15 s and 68° C. for 10 s. After PCR amplification, thePCR products were directly used for Cas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from step 1, 0.25 μM LbCas12a, 1 μL of PCR products, 0.5μM fluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1, and 0.5 μL of RNAenzyme inhibitor. The reaction was carried out at 37° C. for 15 min, andthen stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.13 , the fluorescence response values were gradually lowered with thedecrease of the concentration of E. coli MG1655. Among these,microorganisms were obviously detected in samples 2, 4, 5 and 6.

Example 7. Testing of Human SNP

Five sites of human SNP were selected for SNP testing, including rs5082,rs1467558 , rs2952768 , rs4363657 and rs601338, to test the feasibilityof the HOLMES method.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides (Table 5) were annealed to prepare the template fortranscription. Specifically, 4 μM of paired oligonucleotides wereannealed in 1×PCR buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). crRNA was synthesized using T7 high-yieldtranscription kit, and the reaction was performed at 37° C. overnight(about 16 h). The RNA was purified with RNA Clean & Concentrator ™-5(Zymo Research), quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. PCR amplification: The total volume of reaction system was 20 μL,0.25 μM primers were used (Table 4), 1 ng of human genome (HEK293T) ordirectly scraped oral mucous epithelium was used as the template, andthe high-fidelity polymerase KOD FX (ToYoBo) was used for PCR reaction.The PCR reaction procedure was at 95° C. for 2 min, followed by 35cycles of 98° C. for 10 s, 60° C. for 15 s and 68° C. for 10 s. AfterPCR was completed, they were directly used for Cas12a reaction (Primers,such as 1-rs5082-F-T, 2-rs1467558-F-T and 3-rs2952768-R-C were directlyintroduced into respective mutated products of SNP).

3. Cas12a reaction: In a 20-μL reaction system was added with 1 μMrespective crRNA, 0.5 LbCas12a, 1 μL of PCR products, and 0.5 μMfluorescent probe (HEX-N12-BHQ1). The reaction was carried out at 37° C.for 15 min, and then stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.14 , only with the crRNA corresponding to respective target sequences,higher fluorescence response values were generated, and if one-pointmutation occurred, the response value thereof would be largely reduced.The corresponding SNP genotype could be determined based on thefluorescence value, and these results were confirmed by sequencingresults.

Example 8. Testing of Cancer-Related Genes

TP53 gene was selected for testing gene. TP53 gene contains a nonsensemutation in human T24 cells, which results in the inactivation of thisgene. Cells with gene at this site being normal (HEK293T), individualgene and mutated cell T24 were tested.

1. Preparation of crRNA: First, T7-crRNA-F was annealed with syntheticoligonucleotides of T7-crRNA-34-TP53-T24-C-16 nt andT7-crRNA-34-TP53-T24-G-16 nt (Table 5) to prepare the template fortranscription. Specifically, 4 μM of paired oligonucleotides wereannealed in 1×PCR buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). The crRNA was synthesized using T7high-yield transcription kit, and the reaction was performed at 37° C.overnight (about 16 h). The RNA was purified with RNA Clean &Concentrator™-5 (Zymo Research), quantitated with NanoDrop 2000C,diluted to a concentration of 10 μM and stored at −80° C. in arefrigerator.

2. PCR amplification: The total volume of reaction system was 20 μL,0.25 μM primers of 34-TP53-T24-F and 34-TP53-T24-R were used (Table 4),1 ng of human genome (HEK293T, T24) or directly scraped oral mucousepithelium was used as the template, and the high-fidelity polymeraseKOD FX (ToYoBo) was used for PCR reaction. The PCR reaction procedurewas at 95° C. for 2 min, followed by 35 cycles of 98° C. for 10 s, 60°C. for 15 s and 68° C. for 10 s. After PCR was completed, they weredirectly used for Cas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 1 μMrespective crRNA, 0.5 μM LbCas12a, 1 μL of PCR products, and 0.5 μMfluorescent probe (HEX-N12-BHQ1). The reaction was carried out at 37° C.for 15 min, and then stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.15 , when value of crRNA-C detected by using TP53 gene being normal atthis site as the template was obviously higher than that of crRNA-G,crRNA-G of the mutated cell T24 was obviously increased.

Example 9. Testing of Human SNPs (Gout Related Genes)

Five sites of human SNP were selected for SNP testing, which are relatedto risk of gout, including rs1014290, rs6449213, rs737267, rs1260326 andrs642803, to test the HOLMES method.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides (Table 5) were annealed to prepare the template fortranscription. Specifically, 4 μM of paired oligonucleotides wereannealed in 1×PCR buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). crRNA was synthetized using T7 high-yieldtranscription kit, and the reaction was performed at 37° C. overnight(about 16 h). The RNA was purified with RNA Clean & Concentrator ™-5(Zymo Research), quantitated with NanoDrop 2000C, diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator.

2. PCR amplification: The total volume of reaction system was 20 μL,0.25 μM primers were used (Table 4), 1 ng of human genome (HEK293T) ordirectly scraped oral mucous epithelium was used as the template, andthe high-fidelity polymerase KOD FX (ToYoBo) was used for PCR reaction.The PCR reaction procedure was at 95° C. for 2 min, followed by 35cycles of 98° C. for 10 s, 60° C. for 15 s and 68° C. for 10 s. AfterPCR was completed, they were directly used for Cas12a reaction.(Primers, such as 1-rs5082-F-T, 2-rs1467558-F-T and 3-rs2952768-R-C weredirectly introduced into respective mutated products of SNP).

3. Cas12a reaction: In a 20-μL reaction system was added with 1 μMrespective crRNA, 0.5 μM LbCas12a, 1 μL of PCR products, and 0.5 μMfluorescent probe (HEX-N12-BHQ1). The reaction was carried out at 37° C.for 15 min, and then stopped at 98° C. for 2 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into the 96-well plate and then detected with a microplate readerwith the excitation at 535 nm and emission at 556 nm. As shown in FIG.16 , only with the crRNA corresponding to respective target sequences,higher fluorescence response values were generated, and if one-pointmutation occurred, the response value would be largely reduced. Thecorresponding SNP genotype could be determined based on the fluorescencevalue, and the these results were confirmed by sequencing results.

Example 10. Testing of the Volunteers' Clinic Samples with Kit SNPs(Gout Related Genes)

Premix was added into the 96-well plate to make a kit, then the genomicDNA of 21 volunteers was added to test the rs1014290 site, which isrelated to gout risk.

1. Preparation of kit: First, T7-crRNA-F and synthetic oligonucleotides(Table 5) were annealed to prepare the template for transcription.Specifically, 4 μM of paired oligonucleotides were annealed in 1×PCRbuffer (Transgen Biotech) with a volume of 50-μL, following theannealing procedure: initial denaturation at 95° C. for 5 min, and thencooling down from 95° C. to 20° C. using thermocycler (decrease by 1° C.per min). crRNA was synthetized using T7 high-yield transcription kit,and the reaction was performed at 37° C. overnight (about 16 h). The RNAwas purified with RNA Clean & Concentrator™-5 RNA (Zymo Research),quantitated with NanoDrop 2000C, and diluted to a concentration of 10μM.

2. Premix of PCR reagents in the 96-well plate: Reagents required forPCR were added into the 19-μL system, using primers of 41-rs1014290-Fand 41-rs1014290-R.

3. Premix in the 96-well plate for fluorescence detection: 1 μM crRNA,0.5 μM LbCas12a was added into the 19-μL system and 0.5 μM fluorescentprobe (HEX-N12-BHQ1) was added into the 96-well plate.

4. PCR amplification: The volunteers' genomic DNA was added into abovepremixed PCR 96-well plate for PCR reaction, and the PCR reactionprocedure was at 95° C. for 2 min, followed by 35 cycles of 98° C. for10 s, 60° C. for 15 s and 68° C. for 10 s.

5. Cas12a reaction: 1 μL of PCR reaction liquid was added into thepremixed 96-well plate for fluorescence detection and reaction wasperformed at 37° C. for 15 min and then stopped at 98° C. for 2 min.

6. Fluorescence detection: Detection was performed by a microplatereader with the excitation at 535 nm and emission at 556 nm. As shown inFIG. 17 , population with A: A genotype had higher gout risk, so, thoseexcept volunteer 5, 7 and 9 who had either A: G or G: G genotypes shallpay more attention to gout risk.

Example 11. Detection of Microorganisms Such as E. coli in EnvironmentalWater by LAMP in Combination with Cas Protein

E. coli gyrB gene was chosen as the detection target to indirectly testthe presence and absence of microorganisms such as E. coli in water.

1. Preparation of crRNA: First, T7-crRNA-F and syntheticoligonucleotides of T7-crRNA-gyrB (Table 5) were annealed to prepare thetemplate for transcription. Specifically, 4 μM of pairedoligonucleotides were annealed in 1×Taq DNA polymerase reaction buffer(Transgen Biotech) with a volume of 50-μL, following the annealingprocedure: initial denaturation at 95° C. for 5 min, and then coolingdown from 95° C. to 20° C. using thermocycler (decrease by 1° C. permin). crRNA was synthetized using T7 high-yield transcription kit, andthe reaction was performed at 37° C. overnight (about 16 h). Then, theRNA was purified with an RNA purification and concentration kit,quantitated with NanoDrop 2000C, finally diluted to a concentration of10 μM and stored at −80° C. in a refrigerator for later use.

2. LAMP amplification: Sterilized water and liquid polluted with E. coliwas taken as the negative control and sample to be detected,respectively. The total volume of each reaction system was 25 μL, 1.6 μMLAMP-FIP and LAMP-BIP primers, 0.2 μM LAMP-F3 and LAMP-B3 primers, and0.4 μM LAMP-LoopF and LAMP-LoopB primers were used, and WarmStart® LAMPKit (NEB) was used for LAMP reaction. LAMP reaction procedure was at 65°C. for 30 min. After LAMP was completed, reaction was quenched at 80° C.for 10 min, which were directly used for Cas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from Step 1, 0.25 μM Cas12a, 1 μL of LAMP products, 0.5μM fluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1 and 0.5 μL of RNAenzyme inhibitor. The reaction was carried out at 37° C. for 15 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into a 96-well plate and detected with a microplate reader withthe excitation at 535 nm and emission at 556 nm. The result is shown inFIG. 19 .

Example 12. Detection of SNP with LAMP Amplification in Combination withCas Protein

1. Preparation of crRNA: T7-crRNA-F and synthetic oligonucleotides ofT7-crRNA-rs5082-T/T7-crRNA-rs5082-G/T7-crRNA-rs1467558-T/T7-crRNA-rs1467558-C(Table 5) were annealed to prepare the template for transcription.Specifically, 4 μM of paired oligonucleotides were annealed in 1×Taq DNApolymerase reaction buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). crRNA was synthetized using T7 high-yieldtranscription kit, and the reaction was performed at 37° C. overnight(about 16 h). Then, the RNA was purified with an RNA purification andconcentration kit, quantitated with NanoDrop 2000C, finally diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator for lateruse.

2. LAMP amplification: Human genomic HEK293T was used as the sample. Thetotal volume of each reaction system was 25 μL 1.6 μM LAMP-FIP andLAMP-BIP primers, 0.2 μM LAMP-F3 and LAMP-B3 primers, and 0.4μMLAMP-LoopF and LAMP-LoopB primers were used, and WarmStart® LAMP Kit(NEB) was used for LAMP reaction. LAMP reaction procedure was at 65° C.for 30 min. After LAMP was completed, the reaction was quenched at 80°C. for 10 min, which were directly used for Cas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from Step 1, 0.25 μM Cas12a, 1 μL of LAMP products, 0.5μM fluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1 and 0.5 μL of RNAenzyme inhibitor. The reaction was carried out at 37° C. for 15 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into a 96-well plate and detected with a microplate reader withthe excitation at 535 nm and emission at 556 nm. The result is shown inFIG. 20 .

Example 13. Detection of Microorganisms such as E. coli in EnvironmentalWater by RPA Amplification in Combination with Cas Protein

E. coli gyrB gene was chosen as the detection target to indirectly testthe presence and absence of microorganisms such as E. coli in water.

1. Preparation of crRNA: T7-crRNA-F and synthetic oligonucleotides ofT7-crRNA-gyrB (Table 5) were annealed to prepare the template fortranscription. Specifically, 4 μM of paired oligonucleotides wereannealed in 1×PCR buffer (Transgen Biotech) with a volume of 50-μL,following the annealing procedure: initial denaturation at 95° C. for 5min, and then cooling down from 95° C. to 20° C. using thermocycler(decrease by 1° C. per min). crRNA was synthesized using T7 high-yieldtranscription kit, and the reaction was performed at 37° C. overnight(about 16 h). Then, the RNA was purified with an RNA purification andconcentration kit, quantitated with NanoDrop 2000C, finally diluted to aconcentration of 10 μM and stored at −80° C. in a refrigerator for lateruse.

2. RPA amplification: Sterilized water and liquid polluted with E. coliwas taken as the negative control and sample to be detected,respectively. The total volume of each reaction system was 25 μL, 0.5 μMRPA-gyrB-F (or RPA-gyrB-F2) and RPA-gyrB-R2 were used as primer, andTwistAmp® Basic Kit (TwistDX) was used for RPA reaction. RPA reactionprocedure was at 37° C. for 30 min. After RPA was completed, thereaction was quenched at 80° C. for 10 min, which were directly used forCas12a reaction.

3. Cas12a reaction: In a 20-μL reaction system was added with 0.5 μMcrRNA purified from Step 1, 0.25 μM Cas12a, 1 μL of RPA products, 0.5 μMfluorescent probe (HEX-N12-BHQ1), NEB buffer 3.1 and 0.5 μL of RNAenzyme inhibitor. The reaction was carried out at 37° C. for 15 min.

4. Fluorescence detection: 20 μL of inactivated reaction liquid wasadded into a 96-well plate and detected with a microplate reader withthe excitation at 535 nm and emission at 556 nm. The result is shown inFIG. 21 .

Example 14. Cas12b had Collateral Cleavage Activity

1. Preparation of Guide RNA (sgRNA)

First, the plasmid pUC18-guide RNA-T1 with pUC18 as the plasmid backbonewas constructed by inserting T7 promoter and the template DNA sequencefor transcribing guide RNA into pUC18 (note: the guide RNA transcribedfrom the template was targeted to the sequence named T1 in theresearch). The method is as follow: PUC18 plasmid was used as thetemplate with pUC18-1-F and pUC18-1-R as primers for the first round ofPCR amplification, and the PCR products were ligated using T4 DNA ligasebefore being transformed into DH10b. The correct clone was obtained bysequencing, which was named as pUC18-guide RNA-T1-pre. The pUC18-guideRNA-T1-pre was then used as the template for the second round of PCRamplification with pUC18-2-F and pUC18-2-R as primers. In a similar way,the PCR products were ligated and transformed into DH10b, and finally, acorrectly sequenced plasmid pUC18-guide RNA-T1 is obtained.

Then, guide RNA was synthetized, using T7 high-yield transcription kit(Thermo), with the plasmid PUC18-guide RNA-T1 as the template. Thereaction was performed overnight at 37° C. (12-16 h).

At last, DNase I was added into the transcription system (2 μL of DNase1 added per 50 μL of the transcription system) and the system was put in37° C. water bath for 30 min to remove plasmid DNA. The RNA was thenpurified with an RNA purification and concentration kit, quantitatedwith NanoDrop 2000C, diluted to a concentration of 10 μM and stored at−80° C. in a refrigerator for later use.

2. Preparation of Target DNA:

(1) If the target DNA is single stranded, a 66-bp oligonucleotide wasdirectly synthesized as the target DNA (target-T1-R), in which the 20-bptarget sequence (T1) recognized by guide RNA is contained.

(2) If the target DNA is double stranded, two complementary 66-bpoligonucleotides (i.e. target-T1-F and target-T1-R) were directlysynthesized, in which the 20-bp target sequence (T1) recognized by guideRNA is contained. The two oligonucleotides were annealed to obtaintarget DNA. Specifically, 1 μM of paired oligonucleotides were annealedin 1×PCR buffer (Transgen Biotech) with a volume of 20-μL, following theannealing procedure: initial denaturation at 95° C. for 5 min, and thencooling down from 95° C. to 20° C. using thermocycler (decrease by 1° C.per min).

3. Cas12b Reaction:

(1) Annealing of guide RNA: guide RNA was diluted to an appropriateconcentration (10 μM) and annealed in a PCR instrument, following theannealing procedure: denaturation at 75° C. for 5 min, and then coolingdown from 75° C. to 20° C. (decrease by 1° C. per min).

(2) Incubation of guide RNA and C2c1: the annealed guide RNA and theC2c1 of equal molar concentrations were mixed and placed at 30° C. for20-30 min.

(3) Cas12b reaction: In a 20-μL reaction system, a mixture of guide RNAand C2c1 (the concentration of both is 250 μM or 500 μM) incubated inStep (2), target DNA (final concentration of 50 nM), FAM-labelledoligonucleotides (target-DNMT1-3-R-FAM-5′) or fluorescence quenchingprobe (HEX-N12-BHQ1, final concentration of 500 nM), 2 μL of 10×NEBBuffer 3.1 and 0.5 μL of RNA enzyme inhibitor (40 U/μL) were added.After mixing well, they were allowed to react at 48° C. for 30min.Afterwards, they were heated at 98° C. for 5 min in a PCR instrument forinactivation.

4. Detection of Cas12b for Trans-Cleavage Activity by the UreaDenaturing Gel Electrophoresis Method:

20 μL of inactivated reaction liquid was electrophoretically separatedby the urea denaturing gel electrophoresis method, followed by beingimaged with the fluorescence imaging system ImageQuant LAS 4000 mini (GEHealthcare). The result is shown in FIG. 22 .

5. Detection of Cas12b for Trans-Cleavage Activity by a FluorescenceMicroplate Reader:

20 μL of inactivated reaction liquid was added into a 96-well plate anddetected with a microplate reader with the excitation at 535 nm andemission at 556 nm. The result is as shown in FIG. 23 .

Example 15. Sensitivity Test of the Trans Reaction (Trans-Cleavage) ofCas12b

Based on the detection of the fluorescence intensity excited by thefluorescence probe (HEX-N12-BHQ1), the target DNA concentration requiredfor Cas12b to perform trans-cleavage activity could be determined, whichis the sensitivity of Cas12b trans-cleavage reaction.

1. Preparation of Guide RNA:

First, the 20 bases of the target DNA targeting T1 in the guide RNA weresubstituted with the guide RNA targeting DNMT1-3 to obtain anotherplasmid pUC18-guide RNA-DNMT1-3 by PCR, with pUC18-guide RNA-T1 as thetemplate as well as with the guide RNA-DNMT1-3-F and the guideRNA-DNMT1-3-R as primers.

Second, guide RNA was synthesized using the T7 high-yield transcriptionkit (Thermo), with the plasmid PUC18-guide RNA-DNMT1-3 as the template,the reaction was performed at 37° C. overnight (12-16 h).

At last, DNase I was added into the transcription system (2 μL of DNaseI per 50 μL of the transcription system), and was put in a 37° C. waterbath for 30 min to remove plasmid DNA, the RNA was then purified with anRNA purification and concentration kit, and then quantitated withNanoDrop 2000C, and stored at −80° C. in a refrigerator for later use.

2. Preparation of Target DNA

For the target DNA, the first method was a reaction system directlyadded with the Cas12b without amplification, as follows:

(1) if the target DNA is single stranded, a 50-bp oligonucleotide wasdirectly synthesized as the target DNA (DNMT1-3(TTC PAM)-R), in whichthe 20-bp target sequence (DNMT1-3) recognized by guide RNA iscontained.

(2) if the target DNA is double stranded, two complementary 50-bpoligonucleotides (i.e. DNMT1-3 (TTC PAM)-F and DNMT1-3 (TTC PAM)-R))were directly synthesized, in which the 20-bp target sequence (DNMT1-3)recognized by guide RNA is contained. The two oligonucleotides wereannealed to obtain short target DNA. Specifically, the pairedoligonucleotides (2 μM) were annealed in 1×PCR buffer (Transgen Biotech)of the total volume 20 μL, followed by performing the annealingprocedure: initial denaturation at 95° C. for 5 min, and then coolingdown from 95° C. to 20° C. in a thermocycler (decrease by 1° C. permin).

(3) the single stranded or double stranded target DNA was seriallydiluted to 2 μM, 0.2 μM, 0.02 μM, 0.002 μM and 0.0002 μM for later use.

The second method is that a fragment with the target sequence (DNMT1-3)was inserted into a plasmid vector and amplified by LAMP reaction.

(1) The fragment with the target sequence (DNMT1-3) was inserted intothe pEasy-Blunt Zero Cloning Vector using the pEasy-Blunt Zero CloningKit of Transgen company and verified by sequencing to obtain correctcloning.

(2) LAMP Amplification Reaction

The above plasmids were used as the template for LAMP amplificationreaction, and the templates were added with 0 nM, 1 nM, 0.1 nM (dilutedto 10⁻¹¹ nM at 10 fold gradient). The total volume of each reactionsystem was 25-μL, the primers used is 1.6 μM LAMP-DNM-FIP andLAMP-DNM-BIP primers, 0.2 μM LAMP-DNM-F3 and LAMP-DNM-B3 primers, and0.4 μM LAMP-DNM-LoopF and LAMP-DNM-LoopB, the kit used in the LAMPreaction was WarmStart® LAMP Kit (NEB). LAMP reaction program was at 65°C. for 30 min. After LAMP was completed, quenching at 80° C. for 10 min,and then directly used for Cas12b reaction.

3. Cas 12b Reaction

(1) Annealing of guide RNA: guide RNA was diluted to an appropriateconcentration (5 μM), and placed in a PCR instrument for annealing. Theannealing procedure: denaturation at 75° C. for 5 min, and then coolingdown from 75° C. to 20° C. (decrease by 1° C. per min).

(2) Incubation of guide RNA and Cas12b: the annealed guide RNA and theCas12b of equal molar concentration were mixed and placed at 30° C. for20-30 min.

(3) Cas 12b reaction: in a 20-μL reaction system, a mixture of guide RNAand Cas12b (the concentration of both is 250 μM) incubated in Step (2),1 μL of target DNA or 1 μL of LAMP products, fluorescence probe(HEX-N12-BHQ1) (final concentration 500 nM), as well as 2 μL of 10×NEBbuffer 3.1 and 0.5 μL of RNA enzyme inhibitor (40 U/μL) were added.After mixing well, they were allowed to react at 48° C. for 30min.Afterwards, they were heated at 98° C. for 5 min in a PCR instrument toquench.

4. Detection of Cas 12b Trans-Cleavage Activity by a FluorescenceMicroplate Reader:

20 μL of reaction liquid inactivated was added into a 96-well plate anddetected with a microplate reader with the excitation at 535 nm andemission at 556 nm. Upon combining with LAMP amplication, the Cas12bcould produce significant collateral ssDNA trans-cleavage activity foras low as 10 aM target DNA. As is shown in FIG. 24 .

The cis cleavage property of Cas12a to cleave target ssDNA:

First, in order to test the ssDNA cleavage property of Cas12a, severalcrRNAs (Table 1) targeting the short ssDNA (DMNT1-3) were designed with5(6)-carboxyfluorescein (FAM) labelled at the 3′ end. After cleavage ofFnCas 12a, the reaction product was analyzed by denatured ureapolyacrylamide gel electrophoresis (urea PAGE). Single-stranded DNAcleavage by Cas12a was found to be programmable, i.e., the cleavage siteis from the first base in the 3′ end of the target sequence paired withthe crRNA guide sequence to near the 22nd base (from 21st to 23rd bases)of the target sequence counted in the 5′ end, as shown in FIG. 1A and1C. The dsDNA cleavage of Cas12a requires a PAM sequence, whereas ssDNAcleavage does not require a PAM sequence (FIG. 1A, 1B and FIG. 2 ),which is similar to Cas9-mediated ssDNA cleavage. However, theCas12a-mediated ssDNA cleavage activity is dependent on the stem-loopstructure in crRNA, as shown in FIG. 1A, while Cas9 still exhibits weakcleavage activity against ssDNA with only 20-nt complementary RNAsequences. The stem-loop structure of crRNA is important for stabilizingthe structure of Cas12a, which is responsible for the necessity ofstem-loop structure of crRNA for ssDNA cleavage of Cas12a. It is furthertested whether the ssDNA cleavage site by Cas12a can passed through ashorter guide sequence crRNA, such that the cleavage is outside therecognition site. When the length of the guide sequence is 16 nt, 18 ntor 20 nt, all of these crRNAs resulted in cleavage by Cpf1 near the 22ndbase, as shown in FIG. 1B and 1D, meaning that the cleavage site is 4nt, 2 nt or 0 nt outside the recognition site. Next, the cleavageefficiency of Cas12a for different substrates was tested using dsDNA andssDNA substrates, respectively, as shown in FIG. 1F. Similar to the caseof Cas9 cleavage, ssDNA cleavage is slower than dsDNA cleavage, as shownin FIG. 1E to 1G. These results indicate that the mechanism of Cas12a'sssDNA recognition and cleavage may be different from dsDNA, which is aless efficient PAM-independent recognition cleavage mode; the PAMsequence accelerates target dsDNA recognition and/or cleavage by Cas12a.

The trans cleavage property of Cas12a to cleave ssDNA:

When the target ssDNA is labelled at the 3′ end, Cas12 cleaves aroundthe 22nd base, as shown in FIG. 1 . However, when the labelling was atthe 5′ end, no cleavage product band of the predicted size was observed,but a short (<6 nt) FAM-labelled product was produced, as shown in FIG.3B. Through detailed experiments, once the ternary complexCas12a/crRNA/target ssDNA is formed, the 5′-end labelled target ssDNA(DNMT1-3) (Table 1) is cleaved and a short FAM-labelled product isproduced, as shown in FIG. 3C. In addition, the ternary complex alsocleaves ssDNA that does not have any complementary sequence to the crRNAin any other reaction system (i.e., collateral ssDNA), as shown in FIG.3C and 3D. This cleavage phenomenon is a trans cleavage, which isdistinguished from a programmable cis cleavage. Trans cleavage was alsoobserved when the target ssDNA was labelled at the 3′-end, but manycis-cleaved products were left, as shown in FIG. 3B, which may be due tothe formation of complex Cas12a/crRNA/target ssDNA, which protected thelabelled 3′-end of the target ssDNA from being exposed in the activenuclease site of the ternary complex, and these cleavage processes canbe shown in FIG. 3A.

In addition to FnCas12a tested above, nine Cas12as from other speciessource were also tested (Table 2 and FIG. 4A). Except for Lb4Cas12a, allCas12a have high endonuclease activity on plasmid DNA (as shown in FIG.4B), and all Cas12a ternary complexes show both cis and trans cleavageactivity on ssDNA (as shown in FIG. 4C and 4D). This suggests that cisand trans activity of Cas12a against ssDNA is a common phenomenon.

Key sites and mechanisms of the cis and trans cleavage of ssDNA byCas12a

To determine key amino acid residues in Cas12a for cis and transactivity on ssDNA, several candidate residues of Cas12a were mutated forthe activity testing. First, three single amino acid mutants of FnCas12a(H843A, K852A and K869A) were purified and tested, the residues of whichare related to the RNase activity. The results of the ssDNAtrans-activity studies showed that wild-type FnCas12a and three mutantsshowed no significant difference in cis and trans cleavage activityagainst ssDNA, as shown in FIG. 5A and 5C.

Next, when the endonuclease activity site in FnCas12a is mutated, i.e.,the RuvC domain (D917A, E1006A or D1255A) and the Nuc domain (R1218A)site, the ssDNA cis and trans-cleavage activity of these mutant Cas12awere affected as shown in FIG. 5B and 5D. These results indicate thatthe key sites of Cas12a responsible for target dsDNA cleavage areclosely related to the cis and trans cleavage activities of ssDNA.

A recent structural study of Cas12b (i.e. C2c1), including with extendedtarget DNA or extended non-target DNA complexes, revealed that bothstrands were located within the RuvC pocket, as shown in FIG. 6A and 6B.By comparing the endonuclease catalyzed residues of Cas12b (i.e. C2c1)and Cas12a, it is found that these sites most likely to play a similarrole in the cleavage and function of Cas12b (i.e. C2c1)and Cas12a. Theresults of the in vitro single amino acid mutation experiment show thatit is consistent with the above hypothesis, that is, Cas12a is likely tocleave both strands by only one RuvC catalytic pocket.

Trans cleavage activity of the Cas12a complex: In the structure of theCas12b (i.e. C2c1) complex with additional ssDNA, sequence-independentssDNA is also located on the surface of the catalytic pocket, as shownin FIG. 6C, which is similar to the collateral ssDNA substrate forCas12a . Combining single amino acid mutation experiments, it isproposed that target DNA, non-target DNA and collateral ssDNA were allcleaved in a single RuvC pocket in Cas12a, as shown in FIG. 6D, 6E and6F. The reason why the ternary Cas12a complex has collateral ssDNA transcleavage activity but the monomeric or binary complex does not havecollateral ssDNA trans cleavage activity can be explained by comparingmonomeric, binary, and ternary complex structures. The monomeric Cas12astructure is disordered. The binary complex Cas12a/crRNA is a triangularstructure, as shown in FIG. 6G, while the ternary complexCas12a/crRNA/target DNA is converted into a bilobal structure, therebyexposing the catalytic pocket to achieve trans cleavage of thecollateral ssDNA (as shown in FIG. 6H).

Establishment of Nucleic Acid Probing Methods

Using the characteristics of Cas12a, a method for specifically detectingnucleic acid molecules has been developed, which is called HOLMES (oneHour Low-cost Multipurpose Efficient Simple assay). As the name of thetechnology, it is characterized as a one hour, low cost, multi-purpose,efficient, and simple test method.

In the whole reaction system, it can be divided into two large steps.One is the amplification of the template nucleic acid, and the other isthe specific nucleic acid detection of the Cas12a protein. Here, PCR isused for amplification of nucleic acids, but in practice, anyamplification method can be combined with nucleic acid detection of thesecond step, such as isothermal amplification method RPA or the like.The initial nucleic acid is not limited to double-stranded DNA, and maybe single-stranded DNA; even RNA can be detected by reversetranscription, and thus the method is applicable to various types ofnucleic acid molecules. For the nucleic acid detection stage, threecomponents are the key to the experiment, namely Cas12a, crRNA and thenucleic acid probe. In addition to the 10 Cas12a mentioned in theexamples (these 10 proteins are randomly selected), other Cas12aproteins are equally suitable for this method. In addition, other typesof Cas proteins (such as C2c1 protein) are also suitable for the scopeof the present invention. According to experimental results,Alicyclobacillus acidoterrestris Cas12b (i.e. C2c1) also has acollateral single-stranded DNA trans cleavage activity similar toCas12a, its complex with crRNA/target DNA can also cleave collateralsingle-stranded DNA.

The crRNA as a guiding role is more stable in the system after beingengineered by artificial modification or the like. In the selection ofnucleic acid probes, HEX and BHQ1 labelled short single stranded DNA isselected in the present invention, and any other detectable labelingmethod is theoretically applicable as long as producing a detectabledifference after the nucleic acid probe is cleaved. Alternatively, thenucleic acid probe can also be designed to be fluorescent after bindingto the compound to probe whether the probe is cut off.

In addition, it should be understood that, after reading the aboveteachings of the present invention, those skilled in the art may makevarious modifications and changes to the present invention, theseequivalent forms also fall within the scope defined by the claimsappended hereto.

TABLE 1 Cas12a property experiment related cleavage substrate sequenceOligo name Sequence (5′-3′) SEQ ID No.: target-DNMT1-3-Faatgtttcctgatggtccatgtctgttactcgcctgtcaagtggcgt 1 gac target-DNMT1-3-Rgtcacgccacttgacaggcgagtaacagacatggaccatcagg 2 aaacatt target-DNMT1-3-R-gtcacgccacttgacaggcgagtaacagacatggaccatcagg 3 FAM-3′ aaacatt-FAMtarget-DNMT1-3-R- FAM- 4 FAM-5′gtcacgccacttgacaggcgagtaacagacatggaccatcagg aaacatt target-T1-Ftttctgtttgttatcgcaactttctactgaattcaagctttactctagaa 5 agaggagaaaggatcctarget-T1-R ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagt 6tgcgataacaaacagaaa target-T1-F-FAM FAM- 7tttctgtttgttatcgcaactttctactgaattcaagctttactctagaa agaggagaaaggatcctarget-T1-R-FAM ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagt 8tgcgataacaaacagaaa-FAM target-T1-FAM-3′-Ftttctgtttgttatcgcaactttctactgaattcaagctttactctagaa 9agaggagaaaggatcc-FAM target-T1-FAM-5′-R FAM- 10ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagt tgcgataacaaacagaaatarget-DNMT1-3-R- gtcacgccacttgacaggcgagtaacagacatggaccatcagg 11TTT-FAM-3′ TTTcatt-FAM target-DNMT1-3-R-gtcacgccacttgacaggcgagtaacagacatggaccatcagg 12 CCC-FAM-3′ CCCcatt-FAMtarget-DNMT1-3-R- gtcacgccacttgacaggcgagtaacagacatggaccatcagg 13GGG-FAM-3′ GGGcatt-FAM target-DNMT1-3-F-aatgAAAcctgatggtccatgtctgttactcgcctgtcaagtgg 14 AAA cgtgactarget-DNMT1-3-F- aatgGGGcctgatggtccatgtctgttactcgcctgtcaagtgg 15 GGGcgtgac target-DNMT1-3-F- aatgCCCcctgatggtccatgtctgttactcgcctgtcaagtgg 16CCC cgtgac target-T1-1-R acaaacagaaa 17 target-T1-6-R cgataacaaacagaaa18 target-T1-12-R aagttgcgataacaaacagaaa 19 target-T1-18-Ragtagaaagttgcgataacaaacagaaa 20 target-T1-24-Rgaattcagtagaaagttgcgataacaaacagaaa 21 target-T1-24-only-Rgaattcagtagaaagttgcgataa 22 target-T1-18-only-R agtagaaagttgcgataa 23target-T1-12-only-R aagttgcgataa 24 target-T1-6-only-R cgataa 25N25-5FAM FAM- 26 NNNNNNNNNNNNNNNNNNNNNNNNN N25-3FAMNNNNNNNNNNNNNNNNNNNNNNNNN- 27 FAM

TABLE 2 Names and GI numbers of Cas12a protein and Cas12b (i.e. C2c1)protein Name GI number Species FnCas12a 489130501 Francisella tularensisAsCas12a 545612232 Acidaminococcus sp. BV3L6 LbCas12a 917059416Lachnospiraceae bacterium ND2006 Lb5Cas12a 652820612 Lachnospiraceaebacterium NC2008 HkCas12a 491540987 Helcococcus kunzii ATCC 51366OsCas12a 909652572 Oribacterium sp. NK2B42 TsCas12a 972924080Thiomicrospira sp. XS5 BbCas12a 987324269 Bacteroidales bacteriumKA00251 BoCas12a 496509559 Bacteroidetes oral taxon 274 str. F0058Lb4Cas12a 769130406 Lachnospiraceae bacterium MC2017 C2c1 1076761101Alicyclobacillus acidoterrestris

TABLE 3 Plasmid information Plasmids or Strains Relevant properties orgenotypes Sources Plasmids pET28a-TEV pET28a with the thrombin cleavagesite (Carneiro, Silva et al. 2006) changed to the TEV protease cleavagesite pET28a-TEV-FnCas12a pET28a-TEV carrying FnCas12a (Li, Zhao et al.2016) pET28a-TEV-AsCas12a pET28a-TEV carrying AsCas12a (Li, Zhao et al.2016) pET28a-TEV-LbCas12a pET28a-TEV carrying LbCas12a (Lei, Li et al.2017) pET28a-TEV-Lb5Cas12a pET28a-TEV carrying Lb5Cas12a the presentinvention pET28a-TEV-HkCas12a pET28a-TEV carrying HkCas12a the presentinvention pET28a-TEV-OsCas12a pET28a-TEV carrying OsCas12a the presentinvention pET28a-TEV-TsCas12a pET28a-TEV carrying TsCas12a the presentinvention pET28a-TEV-BbCas12a pET28a-TEV carrying BbCas12a the presentinvention pET28a-TEV-BoCas12a pET28a-TEV carrying BoCas12a the presentinvention pET28a-TEV-Lb4Cas12a pET28a-TEV carrying Lb4Cas12a the presentinvention pET28a-TEV-FnCas12a- pET28a-TEV carrying FnCas12a- the presentinvention K869A K869A pET28a-TEV-FnCas12a- pET28a-TEV carrying FnCas12a-the present invention K852A K852A pET28a-TEV-FnCas12a- pET28a-TEVcarrying FnCas12a- the present invention H843A H843ApET28a-TEV-FnCas12a- pET28a-TEV carrying FnCas12a- the present inventionR1218A R1218A pET28a-TEV-FnCas12a- pET28a-TEV carrying FnCas12a- thepresent invention E1006A E1006A pET28a-TEV-FnCas12a- pET28a-TEV carryingFnCas12a- the present invention D917A D917A pET28a-TEV-FnCas12a-pET28a-TEV carrying FnCas12a- the present invention D1255A D1255ApET28a-TEV-C2c1 pET28a-TEV carrying C2c1 the present invention

TABLE 4 Primers used in the HOLMES method test Oligo nameSequence (5′-3′) SEQ ID No.: target-T1-Rggatcctttctcctctttctagagtaaagcttgaattcagtagaaagttg 28 cgataacaaacagaaaM13F-47 cacaattccacacaacatacgagccgga 29 M13R-48tgtagccgtagttaggccaccacttca 30 Target-T1-Fagttttgttatcgcaactttctactgaattc 31 Target-T1-F-1AagttttgAtatcgcaactttctactgaattc 32 Target-T1-F-2AagttttgtAatcgcaactttctactgaattc 33 Target-T1-F-3TagttttgttTtcgcaactttctactgaattc 34 Target-T1-F-4AagttttgttaAcgcaactttctactgaattc 35 Target-T1-F-5GagttttgttatGgcaactttctactgaattc 36 Target-T1-F-6CagttttgttatcCcaactttctactgaattc 37 Target-T1-F-7GagttttgttatcgGaactttctactgaattc 38 Target-T1-F-8TagttttgttatcgcTactttctactgaattc 39 Target-T1-F-9TagttttgttatcgcaTctttctactgaattc 40 Target-T1-F-10GagttttgttatcgcaaGtttctactgaattc 41 Target-T1-AAAN-Faaaagttatcgcaactttctactgaattc 42 Target-T1-F-11AagttttgttatcgcaacAttctactgaattcggtcatag 43 Target-T1-F-12AagttttgttatcgcaactAtctactgaattcggtcatag 44 Target-T1-F-13AagttttgttatcgcaacttActactgaattcggtcatag 45 Target-T1-F-14GagttttgttatcgcaactttGtactgaattcggtcatag 46 Target-T1-F-15AagttttgttatcgcaactttcAactgaattcggtcatag 47 Target-T1-F-16TagttttgttatcgcaactttctTctgaattcggtcatag 48 Target-T1-F-17GagttttgttatcgcaactttctaGtgaattcggtcatag 49 Target-T1-F-18AagttttgttatcgcaactttctacAgaattcggtcatag 50 Target-T1-PAM1A-FagtttAgttatcgcaactttctactgaattc 51 Target-T1-PAM2A-FagttAtgttatcgcaactttctactgaattc 52 Target-T1-PAM3A-FagtAttgttatcgcaactttctactgaattc 53 gyrB-F AGTTGTCGTTCCTCAACTCCGGCGTTTC54 gyrB-R TCGACGCCAATACCGTCTTTTTCAGTGG 55 1-5082-FCTGCCTTTGCTTCTACCTTTGCCTGT 56 1-5082-F-T TTGCTTCTACCTTTGCCTGTTCTGG 571-5082-R TTTTCTGGCTGGGGATGGCCGATGG 58 2-rs1467558-FAGCAATAACACTAATATTGATTCCTTCAG 59 ATATGGACTCCTTTCATAGTA 2-rs1467558-F-TTTGATTCCTTCAGATATGGACTCCTTTCAT 60 AGTATAACG 2-rs1467558-RTGAGCATCGTTATTCTTACGCGTTGTCATT 61 GAAAGAG 3-rs2952768-FAGCCTGGGCAACGAGTGAAACTCTG 62 3-rs2952768-R ACAGGAGGGACAAAGGCCTAAGTGTCC63 3-rs2952768-R-C CATCATAGGATTGGGAAAAGGACATTTCA 64 GTCATTCAG4-rs4363657-F AGAGTCCTTCTTTCTCAATTTTTCAGAATA 65 ATTTAGTACTTTGGGTAC4-rs4363657-R CAGTACTGAAAAAACCTGCCTATCAATAA 66 AAGCCCTAGAC 5-rs601338-FGCTTCACCGGCTACCTTTGCTCCT 67 5-rs601338-R TTCACCTGCAGGCCCCGCAGG 6834-TP53-T24-F CCTGACTTTCAACTCTGTCTCCTTCCTCTT 69 TTTACAGTA 34-TP53-T24-RTGCTGTGACTGCTTGTAGATGGCCATGG 70 41-rs10124290-FAGTTTCCAGACCTCAGTGCACAAGATACT 71 TTTCTAC 41-rs1014290-F-GACCTCAGTGCACAAGATACTTTTCTACGT 72 CATCCAC 41-rs1014290-RAGCTCCAGTGGATGGAAGATCTTTGAGAT 73 CCAG 42-rs6449213-FAGTCAAAGAGATTCATGCCTGGGACTTTA 74 ATCACATTTAT 42-rs6449213-F-CATGCCTGGGACTTTAATCACATTTATCGG 75 AAGG 42-rs6449213-RCAAATCTGTCTCCACCTCTCAGCTCACCTT 76 G 43-rs737267-FTTCTTGAACCCAAACTCACCTGGCATTTA 77 AACTG 43-rs737267-F-AAAACTCACCTGGCATTTAAACTGACTCTG 78 TAAG 43-rs737267-F-TAAACTCACCTGGCATTTAAACTGTCTCTG 79 TAAG 43-rs737267-RTGCCGAGGCTGAGTTCAGCTACTCTCC 80 44-rs1260326-F ACACAGCACCGTGGGTCAGACCTTGC81 44-rs1260326-F-C TGGGTCAGACTTTGCCGGTGAGAGTC 82 44-rs1260326-F-TTGGGTCAGACTTTGCTGGTGAGAGTC 83 44-rs1260326-R AGCAGTGGCCATGTGATGCTGATGATG84 45-rs642803-F CCCCGGCTCTGTTGGCTTTGAGAATTG 85 45-rs642803-F-CCTCTGTTGGCTTTGAGAATTGCCTGTCTGT 86 GTC 45-rs642803-F-TCTCTGTTGGCTTTGAGAATTGTCTGTCTGT 87 GTC 45-rs642803-RACCGATACCTGGCAGCCCTTGGATG 88 HEX-N12-BHQ1 HEX-NNNNNNNNNNNN-BHQ1 89

TABLE 5 Template sequences for transcription of crRNA Oligo nameSequence (5′-3′) SEQ ID No.: T7-crRNA-F GAAATTAATACGACTCACTATAGGG 90T7-T1-24-R gaattcagtagaaagttgcgataaATCTACAACAGTAGAAA 91TTCCCTATAGTGAGTCGTATTAATTTC T7-T1-15-RagaaagttgcgataaATCTACAACAGTAGAAATTCCCT 92 ATAGTGAGTCGTATTAATTTCT7-T1-16-R tagaaagttgcgataaATCTACAACAGTAGAAATTCCCT 93ATAGTGAGTCGTATTAATTTC T7-T1-17-R gtagaaagttgcgataaATCTACAACAGTAGAAATTCCC94 TATAGTGAGTCGTATTAATTTC T7-T1-18-RagtagaaagttgcgataaATCTACAACAGTAGAAATTCC 95 CTATAGTGAGTCGTATTAATTTCT7-crRNA- GAGTAACAGACATGGACCATCAGATCTACAAC 96 DNMT-23 nt-RAGTAGAAATTCCCTATAGTGAGTCGTATTAATT TC T7-crRNA-gacatggaccatcaggaaacattATCTACAACAGTAGAAAT 97 DNMT-(−8)-RTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-aggcgagtaacagacatggaccaATCTACAACAGTAGAAA 98 DNMT-(+4)-RTTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-tgacaggcgagtaacagacatggATCTACAACAGTAGAAA 99 DNMT-(+8)-RTTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-agacatggaccatcagATCTACAACAGTAGAAATTCCC 100 DNMT-16 nt-RTATAGTGAGTCGTATTAATTTC T7-crRNA- acagacatggaccatcagATCTACAACAGTAGAAATTCC101 DNMT-18 nt-R CTATAGTGAGTCGTATTAATTTC T7-crRNA-taacagacatggaccatcagATCTACAACAGTAGAAATTC 102 DNMT-20 nt-RCCTATAGTGAGTCGTATTAATTTC T7-DNMT-(−8)-gacatggaccatcaggaaacattCCCTATAGTGAGTCGTAT 103 no loop-R TAATTTC T7-DNMT-aggcgagtaacagacatggaccaCCCTATAGTGAGTCGTA 104 (+4)-no loop-R TTAATTTCT7-DNMT- tgacaggcgagtaacagacatggCCCTATAGTGAGTCGTAT 105 (+8)-no loop-RTAATTTC T7-crRNA- CCTCTTCCCAGAACAGGATCTACAACAGTAGAA 106 rs5082-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA- CCTCTTCCCAGCACAGGATCTACAACAGTAGAA107 rs5082-G ATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-CTGAAGCGTTATACTATATCTACAACAGTAGAA 108 rs1467558-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA- CTGAAGCGTTGTACTATATCTACAACAGTAGAA109 rs1467558-C ATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-TTTTATCTGAATGATTATCTACAACAGTAGAAAT 110 rs2952768-T-TCCCTATAGTGAGTCGTATTAATTTC 16 nt T7-crRNA-TTTTATCTGAATGACTATCTACAACAGTAGAAAT 111 rs2952768-C-TCCCTATAGTGAGTCGTATTAATTTC 16 nt T7-crRNA-AAAAAAGAGTGAGTACCATCTACAACAGTAGAA 112 rs4363657-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA- AAAAAAGAGTGGGTACCATCTACAACAGTAGAA113 rs4363657-C ATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-GGTAGAAGGTCCAGGAGATCTACAACAGTAGAA 114 rs601338-GATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA- GGTAGAAGGTCTAGGAGATCTACAACAGTAGAA115 rs601338-A ATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-34-GGGCAGGGGAGTACTGATCTACAACAGTAGAAA 116 TP53-T24-C-16 ntTTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-34-GGGCAGGGGACTACTGATCTACAACAGTAGAAA 117 TP53-T24-G-TTCCCTATAGTGAGTCGTATTAATTTC 16 nt T7-crRNA-41-TCAGTGGATGATGTAATCTACAACAGTAGAAAT 118 rs1014290-A-TCCCTATAGTGAGTCGTATTAATTTC 15 nt T7-crRNA-41-TCAGTGGATGACGTAATCTACAACAGTAGAAAT 119 rs1014290-G-TCCCTATAGTGAGTCGTATTAATTTC 15 nt T7-crRNA-42-GGAAATTCTCCTTCCGAATCTACAACAGTAGAA 120 rs6449213-CATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-42-GGAAATTCTCCTTCCAAATCTACAACAGTAGAA 121 rs6449213-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-43-TCTTACAGAGTCAGTTATCTACAACAGTAGAAAT 122 rs737267-A-16 ntTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-43-TCTTACAGAGCCAGTTATCTACAACAGTAGAAA 123 rs737267-G-16 ntTTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-43-GTCTTACAGAGACAGTTATCTACAACAGTAGAA 124 rs737267-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-44-CTGGACTCTCACCGGATCTACAACAGTAGAAAT 125 rs1260326-C-TCCCTATAGTGAGTCGTATTAATTTC 15 nt T7-crRNA-44-CTGGACTCTCACCAGATCTACAACAGTAGAAAT 126 rs1260326-T-TCCCTATAGTGAGTCGTATTAATTTC 15 nt T7-crRNA-45-CACAGACAGGCAATTCTATCTACAACAGTAGAA 127 rs642803-CATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA-45-CACAGACAGACAATTCTATCTACAACAGTAGAA 128 rs642803-TATTCCCTATAGTGAGTCGTATTAATTTC T7-crRNA- gyrBTCGCGCTTGTCGCGCAGACGAATGATCTACAAC 129 AGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC

Primers used in the detection of DNA LAMP amplification in combinationwith Cas12a:

TABLE 6 Primers for amplifying gyrB-1 SEQ ID name sequence No.:LAMP-gyrB-1-F3 CATGGTGCGTTTCTGGCC 130 LAMP-gyrB-1-B3CGGCGTTTTGTTCTTGTTCA 131 LAMP-gyrB-1-FIP ACAACTCACGCAGACGTTTCGCAAC 132CTTCACCAATGTGACCG LAMP-gyrB-1-BIP GTTCCTCAACTCCGGCGTTTCCGAT 133GCCGCCTTCATAGTGG LAMP-gyrB-1-LoopF CAGAATTTCATATTCGAACT 134LAMP-gyrB-1-LoopB GACGGCAAAGAAGACCACTT 135

TABLE 7 Primers for amplifying gyrB-2 SEQ ID name sequence No.:LAMP-gyrB-2-F3 CGACGGCAAAGAAGACCA 136 LAMP-gyrB-2-B3 AGCCTGCCAGGTGAGTAC137 LAMP-gyrB-2-FIP CGGGTGGATCGGCGTTTTGTTCACT 138 ATGAAGGCGGCATCALAMP-gyrB-2-BIP GTATTGGCGTCGAAGTGGCGTTCGC 139 TGCGGAATGTTGTTGLAMP-gyrB-2-LoopF TTGTTCAGATATTCAACGAACG 140 LAMP-gyrB-2-LoopBGTGGAACGATGGCTTCCAGG 141

TABLE 8 Primers for amplifying the rs1467558 site SEQ ID name sequenceNo.: LAMP-rs1467558-F3 CAGCTGTAGACCATAAGCC 142 LAMP-rs1467558-B3GTGGCTGAGCATCGTTAT 143 LAMP-rs1467558-FIP ACTATGAAAGGAGTCCATATCTGAA 144GGAATTCAGGTAGTGGTTTGGGA LAMP-rs1467558-BIP GCTTCAGCCTACTGCAAATCCTACG 145CGTTGTCATTGAAAG LAMP-rs1467558- TCAATATTAGTGTTATTGCTTG 146 LoopFLAMP-rs1467558- TGGTGGAAGATTTGGACAGGAC 147 LoopB

TABLE 9 Primers for amplifying the rs5082 site SEQ ID name sequence No.:LAMP-rs5082-F3 GCTGGAAAGGTCAAGGGAC 148 LAMP-rs5082-B3GGGGTTTGTTGCACAGTCC 149 LAMP-rs5082-FIP CAAAGGTAGAAGCAAAGGCAGGAG 150GTTTGCCCAAGGTCACACAG LAMP-rs5082-BIP CTGGGAAGAGGGAGGGCTCAGTGT 151TGCCACACTTTCACTGG LAMP-rs5082-LoopF GTGAGCGGGTGGGGTGCT 152LAMP-rs5082-LoopB TCTAAGTCTTCCAGCACGGGATC 153

TABLE 10 Primers used in the detection of RPA amplificationin combination with Cas12 SEQ ID name sequence No.: RPA-gyrB-1-FATATGAAATTCTGGCGAAACGTCTGCGTGA 154 GTTG RPA-gyrB-2-FAAACGTCTGCGTGAGTTGTCGTTCCTCAAC 155 TCC RPA-gyrB-RACTTCGACGCCAATACCGTCTTTTTCAGTG 156 GAG

TABLE 1 Primers used in determination Cas12b for transcleavage activity: SEQ ID Oligo name Sequence (5′-3′) No.: pUC18-1-FATCTGAGAAGTGGCACTTATCGCAACTTTCTA 157 CTGAGGTCATAGCTGTTTCCTGTGTGApUC18-1-R GTCCTCTAGACCCCTATAGTGAGTCGTATTAA 158TTTCATGATTACGAATTCGAGCTCGGT pUC18-2-F CCACTTTCCAGGTGGCAAAGCCCGTTGAGCTT159 CTCAAATCTGAGAAGTGGCACTTATC pUC18-2-R TGGAAAGTGGCCATTGGCACACCCGTTGAAA160 AATTCTGTCCTCTAGACCCCTATAGTGA T7-crRNA-F GAAATTAATACGACTCACTATAGGG161 ZL-sgRNA- TCAGTAGAAAGTTGCGATAAGTGC 162 T1-R ZLsgRNA-AACAGACATGGACCATCAGGGTG 163 DNMT1-3-R target-T1-FTTTCTGTTTGTTATCGCAACTTTCTACTGAATT 164 CAAGCTTTACTCTAGAAAGAGGAGAAAGGAT CCtarget-T1-R GGATCCTTTCTCCTCTTTCTAGAGTAAAGCTT 165GAATTCAGTAGAAAGTTGCGATAACAAACAG AAA target-GTCACGCCACTTGACAGGCGAGTAACAGACA 166 DNMT1-3- TGGACCATCAGGAAACATTR-FAM-5′ target-T1-R GGATCCTTTCTCCTCTTTCTAGAGTAAAGCTT 167GAATTCAGTAGAAAGTTGCGATAACAAACAG AAA target-T1-FTTTCTGTTTGTTATCGCAACTTTCTACTGAATT 168 CAAGCTTTACTCTAGAAAGAGGAGAAAGGAT CC

TABLE 12Primers used in the sensitivity test of the trans reaction of Cas12b:Oligo name Sequence (5′-3′) SEQ ID No.: sgRNA-DNMT1-3-FCCTGATGGTCCATGTCTGTTGGTCATAG 169 CTGTTTCCTGTGTG sgRNA-DNMT1-3-RTGGACCATCAGGGTGCCACTTCTCAGAT 170 TTGAGAAG T7-crRNA-FGAAATTAATACGACTCACTATAGGG 171 ZLsgRNA-DNMT1-3-R AACAGACATGGACCATCAGGGTG172 DNMT1-3(TTC PAM)-F AATGTTCCCTGATGGTCCATGTCTGTTA 173CTCGCCTGTCAAGTGGCGTGAC DNMT1-3(TTC PAM)-R GTCACGCCACTTGACAGGCGAGTAACA174 GACATGGACCATCAGGGAACATT LAMP-DNM-F3 gtgaacgttcccttagcact 175LAMP-DNM-B3 gggagggcagaactagtcc 176 LAMP-DNM-FIPcgccacttgacaggcgagtaactgccacttattgggtcagc 177 LAMP-DNM-BIPgcgtgttccccagagtgacttagcagcttcctcctcctt 178 LAMP-DNM-LoopFaggaaacattaacgtactgatg 179 LAMP-DNM-LoopB ttccttttatttcccttcagc 180DNMT1-3(TTC PAM)-R GTCACGCCACTTGACAGGCGAGTAACA 181GACATGGACCATCAGGGAACATT DNMT1-3(TTC PAM)-F AATGTTCCCTGATGGTCCATGTCTGTTA182 CTCGCCTGTCAAGTGGCGTGAC

TABLE 13 Other sequences involved in the present invention Name SequenceSEQ ID No.: AacCas12b sgRNA GTCTAGAGGACAGAATTTTTCAACGGGTGTG 183 sequenceCCAATGGCCACTTTCCAGGTGGCAAAGCCCG TTGAGCTTCTCAAATCTGAGAAGTGGCACcctgatggtccatgtctgtt Guide sequence cctgatggtccatgtctgtt 184 targetingtargetDNMT-1-3 Single-strandedgtcacgccacttgacaggcgagtaacagacatggaccatcagggaacatt 185 target sequenceDouble-stranded gtcacgccacttgacaggcgagtaacagacatggaccatcagggaacatt 186target sequence: amino acid MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNA 187sequence of GVRYYTEWLSLLRQENLYRRSPNGDGEQECDK AacCas12b proteinTAEECKAELLERLRARQVENGHRGPAGSDDEL LQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPG WEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMF QQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESK EQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQ ALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGE RRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGA KIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKL SDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGND NLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKL IEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWR KDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHI DHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQ LMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPW WLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQI RLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEA ELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENT GDI*

All publications mentioned in the present invention are incorporated byreference as if each individual publication was cited as a reference inthe present application. It should be understood that, after reading theabove teachings of the present invention, those skilled in the art canmake various modifications and changes to the present invention. Theseequivalent forms are also within the scope defined by the claimsappended hereto.

What is claimed is:
 1. A method for detecting target nucleic acidmolecules comprising steps of: adding a guide RNA, a Cas protein, anucleic acid probe and a buffer into a system containing the targetnucleic acid molecules to be detected, and then detecting cleavage ofthe nucleic acid probe.
 2. The method of claim 1, wherein the Casprotein has collateral ssDNA cleavage activity and the nucleic acidprobe comprises ssDNA.
 3. The method of claim 1, wherein the guide RNAspecifically binds to the target nucleic acid molecule(s).
 4. The methodof claim 1, wherein the nucleic acid probe comprises adetectably-labeled single-stranded DNA.
 5. The method of claim 1,wherein the target nucleic acid molecules to be detected have beenamplified.
 6. (canceled)
 7. The method of claim 1, characterized in thatthe Cas protein is a Cas12 protein.
 8. (canceled)
 9. (canceled)
 10. Akit for detecting target nucleic acid molecules, characterized in thatthe kit comprises a guide RNA, a Cas protein, and a nucleic acid probe.11. A detection system comprising: (a) target nucleic acid molecules;(b) a Cas protein comprising collateral single-stranded DNA cleavageactivity; (c) a guide RNA, which directs the Cas protein to specificallybind to the target nucleic acid molecules; (d) a nucleic acid probe,which comprises a single-stranded DNA; and (e) buffer wherein the targetnucleic acid molecule is a target DNA.
 12. The detection system of claim11, wherein the Cas protein is selected from the group consisting of:FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a,BbCas12a, BoCas12a, Lb4Cas12a; LbCas12a and Cas12b (i.e., C2c1).
 13. Thedetection system of claim 11, wherein the nucleic acid probe comprises adetectable label.
 14. A kit for detecting target nucleic acid molecules,characterized in that the kit comprises: i) a first container and a Casprotein located in the first container, wherein the Cas protein hascollateral single-strand DNA cleavage activity; ii) a second containerand a guide RNA located in the second container, wherein the guide RNAdirects the Cas protein to specifically bind to the target nucleic acidmolecules; iii) a third container and a nucleic acid probe located inthe third container; and iv) a fourth container and a buffer located inthe fourth container; wherein the target nucleic acid molecule is atarget DNA. 15.-21. (canceled)
 22. The method of claim 1, whereindetecting cleavage of the nucleic acid probe comprises a fluorescencedetection method.
 23. The method of claim 22, wherein the fluorescencedetection method comprises a detection method using a microplate readeror a fluorescence spectrophotometer.
 24. The method of claim 4, whereinthe detectable label is a fluorescent label.
 25. The method of claim 24,wherein the fluorescent label comprises a fluorescent group HEX at the5′ end and a quenching group BHQ1 at the 3′ end.
 26. The method of claim7, wherein the Cas12 protein is selected from the group consisting of:FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a,BbCas12a, BoCas12a, Lb4Cas12a; LbCas12a and Cas12b (i.e., C2c1).
 27. Thedetection system of claim 11, wherein the target nucleic acid moleculesare amplified.
 28. The detection system of claim 27, wherein the targetnucleic acid molecules are amplified by a method selected from the groupconsisting of: PCR amplification, LAMP amplification, RPA amplification,ligase chain reaction, branched DNA amplification, NASBA, SDA,transcription-mediated amplification, rolling circle amplification, HDA,SPIA, NEAR, TMA and SMAP2.
 29. The detection system of claim 27, whereinthe target nucleic acid molecules are amplified using primers comprisingPAM sequence.
 30. The detection system of claim 29, wherein theamplification product comprises a PAM sequence upstream or downstream ofa target site of the guide RNA.
 31. The detection system of claim 30,wherein the PAM site is −20 nt to +20 nt upstream or downstream of thetarget site of the guide RNA
 32. The detection system of claim 11,wherein the target nucleic acid molecules comprise a sequence found in apathogen.
 33. The detection system of claim 11, wherein the targetnucleic acid molecules comprise a sequence found in a human cancer.